Methods for increasing the production of ethanol from microbial fermentation

ABSTRACT

A stable continuous method for producing ethanol from the anaerobic bacterial fermentation of a gaseous substrate containing at least one reducing gas involves culturing in a fermentation bioreactor anaerobic, acetogenic bacteria in a liquid nutrient medium; supplying the gaseous substrate to the bioreactor; and manipulating the bacteria in the bioreactor by reducing the redox potential, or increasing the NAD(P)H TO NAD(P) ratio, in the fermentation broth after the bacteria achieves a steady state and stable cell concentration in the bioreactor. The free acetic acid concentration in the bioreactor is maintained at less than 5 g/L free acid. This method allows ethanol to be produced in the fermentation broth in the bioreactor at a productivity of greater than 10 g/L per day. Both ethanol and acetate are produced in a ratio of ethanol to acetate ranging from 1:1 to 20:1.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/876,312, filed Oct. 22, 2007, which is a continuation of U.S. patentapplication Ser. No. 10/311,655, filed Mar. 11, 2003, now U.S. Pat. No.7,285,402, which is a U.S. national phase of International PatentApplication No. PCT/US01/23149, filed Jul. 23, 2001, which claims thebenefit of the priority of U.S. Provisional Patent Application No.60/220,794, filed Jul. 25, 2000, now abandoned, all of which areincorporated herein their entirety by reference.

BACKGROUND

The present invention is directed to improvements in microbialfermentation methods for the production of ethanol from a gaseoussubstrate containing at least one reducing gas using anaerobic (orfacultative) acetogenic bacteria.

Methods for producing ethanol, among other organic acids, alcohols,hydrogen and organic acid salts, from the microbial fermentation ofgaseous substrates in media containing suitable nutrients and traceminerals using certain anaerobic bacteria have been disclosed by theseinventors. For example, the inventors have previously disclosed thatdilute gas mixtures are introduced into a bioreactor containing one ormore strains of anaerobic bacteria that utilize the waste gas componentsby a direct pathway to produce a desired compound. The compound isrecovered from the aqueous phase in a separate vessel or vessels,utilizing a suitable recovery method for the compound produced. Examplesof recovery methods include extraction, distillation or combinationsthereof, or other efficient recovery methods. The bacteria can beremoved from the aqueous phase and recycled to the bioreactor tomaintain high cell concentrations, thus maximizing productivity. Cellseparation, if desired, is accomplished by centrifugation, membranousfiltration, or other techniques. See, for example, International PatentPublication No. WO98/00558, published Jan. 8, 1998; U.S. Pat. No.5,807,722; U.S. Pat. No. 5,593,886 and U.S. Pat. No. 5,821,111.

In addition to its major product, acetic acid, strains of the anaerobicbacterium Clostridium ljungdahlii are able to also produce ethanol as aproduct in the conversion of carbon monoxide (CO), hydrogen (H₂) andcarbon dioxide (CO₂). The production of acetic acid (CH₃COOH) andethanol (C₂H₅OH) from CO, CO₂ and H₂ are shown by the following overallstoichiometric equations:4CO+2H₂O→CH₃COOH+2CO₂  (1)4H₂+2CO₂→CH₃COOH+2H₂O  (2)6CO+3H₂O→C₂H₅OH+4CO₂  (3)6H₂+2CO₂→C₂H₅OH+3H₂O  (4)

Several exemplary strains of C. ljungdahlii include strain PETC (U.S.Pat. No. 5,173,429); strain ERI-2 (U.S. Pat. No. 5,593,886) and strainsC-01 and O-52 (U.S. Pat. No. 6,136,577). These strains are eachdeposited in the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, under Accession Nos.: 55383(formerly ATCC No. 49587), 55380, 55988, and 55989 respectively. Each ofthe strains of C. ljungdahlii is an anaerobic, gram-positive bacteriumwith a guanine and cytosine (G+C) nucleotide content of about 22 mole %.These bacteria use a variety of substrates for growth, but not methanolor lactate. These strains differ in their CO tolerance, specific gasuptake rates and specific productivities. In the “wild” strains found innature, very little ethanol production is noted. Strains of C.ljungdahlii operate ideally at 37° C., and typically produce an ethanolto acetyl (i.e. which refers to both free or molecular acetic acid andacetate salts) product ratio of about 1:20 (1 part ethanol per 20 partsacetyl) in the “wild” state. Ethanol concentrations are typically only1-2 g/L. While this ability to produce ethanol is of interest, becauseof low ethanol productivity the “wild” bacteria cannot be used toeconomically produce ethanol on a commercial basis with minor nutrientmanipulation the above-mentioned C. ljungdahlii strains have been usedto produce ethanol and acetyl with a product ratio of 1:1 (equal partsethanol and acetyl), but the ethanol concentration is less than 10 g/L,a level that results in low productivity, below 10 g/L·day. In additionculture stability is an issue, primarily due to the relatively high(8-10 g/L) concentration of acetyl (2.5-3 g/L molecular acetic acid) incombination with the presence of ethanol. Furthermore, as the gas rateis increased in an effort to produce more ethanol, the culture isinhibited, first by molecular acetic acid and then by CO. As a result,the culture becomes unstable and fails to uptake gas and produceadditional product. Further, early work by the inventors showeddifficulty in producing more than a 2:1 ratio of ethanol to acetyl in asteady state operation. See, e.g., Klasson et al., 1990 AppliedBiochemistry and Biotechnology, Proceedings of the 11^(th) Symposium onBiotechnology for Fuels and Chemicals, 24/25: 857; Phillips et al., 1993Applied Biochemistry and Biotechnology, Proceedings of the 14^(th)Symposium on Biotechnology for Fuels and Chemicals, 39/40: 559, amongothers.

A large number of documents describe the use of anaerobic bacteria,other than C. ljungdahlii, in the fermentation of sugars that do notconsume CO, CO₂ and H₂ to produce solvents. In an attempt to providehigh yields of ethanol, a variety of parameters have been altered whichinclude: nutrient types, microorganism, specific addition of reducingagents, pH variations, and the addition of exogenous gases. See, e.g.,Rothstein et al, 1986 J. Bacterial., 165(1):319-320; Lovitt et al, 1988J. Bacterial., 170(6):2809; Taherzadeh et al, 1996 Appl. Microbiol.Biotechnol., 46:176.

There remains a need in the art of the handling of industrial gaseoussubstrates, the ability to extract valuable commodities from such gases,particularly waste gases, such as H₂, CO and CO₂. There is a need toenhance the production of ethanol relative to the production of theother products normally generated by the fermentation of such gases byacetogenic bacteria.

SUMMARY

In response to the need in the art, the present invention provides novelmethods which are continuous, steady state methods and which result inethanol concentrations greater than 10 g/L and acetate concentrationslower than about 8-10 g/L, while continuing to permit culture growth andgood culture stability.

In one aspect, the invention provides a stable continuous method forproducing ethanol from the anaerobic bacterial fermentation of a gaseoussubstrate. The method comprising the steps of culturing in afermentation bioreactor anaerobic, acetogenic bacteria in a liquidnutrient medium and supplying to the bioreactor the gaseous substratecomprising at least one reducing gas selected from the group consistingof CO and H₂. The bacteria in the bioreactor are manipulated by reducingthe redox potential, or increasing the NAD(P)H TO NAD(P) ratio, in thefermentation broth after the bacteria achieves a steady state, e.g., astable cell concentration, in the bioreactor. The free acetic acidconcentration in the bioreactor is maintained at less than 5 g/L freeacid. The culturing and manipulating steps cause the bacteria in thebioreactor to produce ethanol in a fermentation broth at a productivitygreater than 10 g/L per day. Both ethanol and acetate are produced inthe fermentation broth in a ratio of ethanol to acetate ranging from 1:1to 20:1.

In one embodiment of this method, the manipulating step includes one ormore of the following steps: altering at least one parameter selectedfrom the group consisting of nutrient medium contents, nutrient feedrate, aqueous feed rate, operating pressure, operating pH, gaseoussubstrate contents, gas feed rate, fermentation broth agitation rate,product inhibition step, cell density, and substrate inhibition.

In another embodiment of this method, the manipulating step comprisessupplying to the bioreactor said gaseous substrate comprising thereducing gas, CO, at a desired rate of uptake. This rate is desirablyfrom 0.3 to 2 mmol CO/gram of dry cell of bacteria in saidbioreactor/minute.

In still another embodiment of this method the manipulating stepcomprises feeding into said fermentation bioreactor said nutrient mediumcomprising a limiting amount of calcium pantothenate. The calciumpantothenate is desirably in a range of from 0.5 to 50 μg/grams of drycell of bacteria produced in the bioreactor.

Another embodiment of the method includes supplying excess H₂ reducinggas to said bioreactor prior to providing the limiting amount of calciumpantothenate.

In yet a further aspect, the invention provides a method in which themanipulating step of the method includes feeding into said fermentationbioreactor said nutrient medium comprising a limiting amount of cobalt.Desirably, the amount of cobalt is in a range of from 5 to 100 μgcobalt/grams of dry cell of bacteria produced in said bioreactor.

In another embodiment, the method of the invention includes preventingacclimation of said bacteria in said bioreactor to said amount of cobaltby maintaining a constant cobalt concentration and adjusting one or moreparameters, such as gas rate, liquid rate, agitation rate and H₂ gaspartial pressure.

Additional optional steps of these methods include subjecting a sampleof the broth to centrifugation to eliminate cells and to gaschromatography to monitor the maintenance of the ratio and/orproductivity values.

In another embodiment, the method comprises feeding as the gaseoussubstrate an amount of H₂ in slight excess of the stoichiometric amountfor ethanol production. In still another embodiment, the gaseoussubstrate further comprises an amount of CO in slight excess of theamounts required by the bacteria, wherein uptake of H₂ by the bacteriais inhibited and the NAD(P)H to NAD(P) ratio in the broth is increased.

In yet another embodiment of the method, a step is provided in whichinhibition by molecular acetic acid is reduced by increasing the aqueousfeed rate when the molecular acetic acid present in the fermentationbroth approaches or exceeds 2 g/L.

In another embodiment of the method, the manipulating step may includeagitating the medium, bacteria and gaseous substrate in the bioreactorat a selected agitation rate. For example, reduction in the agitationrate reduces the amount of CO transferred to the fermentation broth.This reduction in the rate of CO transfer causes an increase in H₂conversion, so that the reducing gas, H₂, is present in the bioreactorin excess of the growth requirements of the bacteria. The gas rate mayalso be similarly reduced to decrease the amount of CO transferred,thereby increasing H₂ conversion, so that the reducing gas, H₂, ispresent in the fermentation bioreactor in excess of the growthrequirements of the bacteria.

In still another embodiment of the method, the bacterial culture mayinitially be brought to the desired cell concentration in the bioreactorbefore limiting the calcium pantothenate or cobalt concentration of thenutrient medium.

In another embodiment of the method of this invention, a two stage CSTR(bioreactor) is used which consists of a growth reactor which feeds thefermentation broth to a production reactor in which most of the ethanolis produced.

In another aspect of the invention, the method described above includesthe optional steps of: recovering ethanol by removing the fermentationbroth from the bioreactor; distilling ethanol from the broth; andrecovering the ethanol. Additionally or preferably, a sample of thebroth is subjected to centrifugation to eliminate cells; and themaintenance of the ratio is monitored using gas chromatography.

In still another aspect, the method of the invention may further employan additional step of recycling water (containing up to 5 g/L acetyl)from the ethanol production back to the reactor so that an equilibriumis established between the ethanol and acetyl in the reactor. As aresult, more of the CO, CO₂ and H₂ fed to the reactor and converted toproducts results in ethanol production.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating a continuous fermentationmethod with product recovery according to this invention. Gaseoussubstrate 1 and liquid phase nutrient medium 2 are fed to bioreactor 3containing the subject bacterial culture. Conversion of the gaseoussubstrate to ethanol and acetic acid takes place in the bioreactor 3.Exhaust gas 4 containing gases other than CO, CO₂ and H₂ and unconvertedCO, CO₂ and H₂ from bioreactor 3 are vented, combusted as fuel orflared. With cell recycle, liquid effluent 5 is sent to cell separator 6where the cells 7 and cell-free permeate 8 are separated. Cells 7 aresent back to bioreactor 3 and permeate 8 is sent to product recovery.Ethanol can be recovered from the permeate 8 (or alternatively from theeffluent 5 if cell separation is not employed). Permeate 8 is separatedin distillation column 9 to produce 95% ethanol overhead 10, and water11 for recycle back to bioreactor 3. The 95% ethanol overhead 10 is sentto a molecular sieve 12 where anhydrous ethanol 13, the desired finalproduct, is separated from dilute ethanol 14 which is sent back to thedistillation column 9.

FIG. 2 is a schematic diagram of a two-stage, continuously stirredreactor (CSTR) system for improved culture stability. Growth stage CSTR1 is fed liquid medium 2. Unconverted gas 3 from the Production StageCSTR is fed to Growth Stage CSTR 1. Production Stage CSTR 4 is fed afresh gas feed 5, and fresh medium feed 6 as well as culture feed 7 fromGrowth Stage CSTR 1. Cell recycle 8 is used to get the most productionout of the cells 9 sent to Production Stage CSTR 4. Cells 9 are notrecycled to the Growth Stage CSTR. Liquid Product 10 consisting ofdilute ethanol in the fermentation broth is produced as the finaldistillation product, and is recovered as anhydrous ethanol as in FIG.1.

DETAILED DESCRIPTION

The present invention involves methods for the anaerobic fermentation ofgaseous substrates containing at least one reducing gas, particularlythe gaseous components of industrial waste and synthesis gases (e.g.,CO, CO₂ and H₂) to ethanol. These methods yield ethanol productivitiesgreater than 10 g/L·day by manipulating the biological pathways of thesubject bacteria. One method of the invention causes an abundance ofNAD(P)H over NAD(P). The oxidation of NAD(P)H to NAD(P) causes aceticacid produced by the culture to be reduced to ethanol. Alternatively,other methods for the production of high concentrations of ethanol in ananaerobic fermentation of this invention involve reducing the redoxpotential of the fermentation broth, and thereby reducing acetic acid toethanol. The methods of this invention produce high ethanolconcentrations (i.e., greater than about 10 g/L, and preferably greaterthan about 15 g/L) and low acetate concentrations (i.e. less than about5 g/L free acetic acid in the bioreactor). These methods also maintainand control method conditions for continuous ethanol and acetic acidproduction to help the system recover rapidly from method upsets.Further, the methods of this invention help prevent culture acclimationto low nutrient concentration, which can be detrimental to cultureperformance. The present invention provides a viable commercial methodfor ethanol production.

I. Definitions

Unless otherwise defined, the following terms as used throughout thisspecification are defined as follows.

The term “continuous method” as used herein refers to a fermentationmethod which includes continuous nutrient feed, substrate feed, cellproduction in the bioreactor, cell removal (or purge) from thebioreactor, and product removal. This continuous feeds, removals or cellproduction may occur in the same or in different streams. A continuousprocess results in the achievement of a steady state within thebioreactor. By “steady state” is meant that all of these measurablevariables (i.e., feed rates, substrate and nutrient concentrationsmaintained in the bioreactor, cell concentration in the bioreactor andcell removal from the bioreactor, product removal from the bioreactor,as well as conditional variables such as temperatures and pressures) areconstant over time.

The term “gaseous substrates” as used herein means CO alone, CO and H₂,CO₂ and H₂, or CO, CO₂ and H₂, optionally mixed with other elements orcompounds, including nitrogen and methane in a gaseous state. Suchgaseous substrates include gases or streams, which are typicallyreleased or exhausted to the atmosphere either directly or throughcombustion. In some embodiments of this method the gaseous substratecomprises CO. In other embodiments of this method, the gaseous substratecomprises CO₂ and H₂. In still other embodiments, the gaseous substratecomprises CO and H₂. In a particularly preferred embodiment, the gaseoussubstrate comprises CO, CO₂ and H₂. Still other substrates of theinvention may include those components mentioned above and at least onegas of nitrogen, CO₂, ethane and methane. Thus, such substrates includewhat is conventionally referred to as “syngas” or synthesis gas from thegasification of carbon products (including methane), as well as wastegases from a variety of industrial methods.

The term “reducing gas” means either or both CO or H₂. By the phrase “anamount of reducing gas greater than that required for growth of thebacteria” means that amount of reducing gas that exceeds the amount thatthe bacteria can use for growth or metabolism, given the nutrient mediumingredients. This amount can be achieved by increasing the net amount ofreducing gas, or by reducing key nutrient ingredients, so that theexcess amount of gas is achieved without increasing the gas, or byincreasing the rate of gas delivery to the bacteria. When the bacteriaare exposed to more reducing gas than required for growth, the bacteriarespond by increasing the producing of ethanol.

“Subject bacteria” are acetogenic anaerobic (or facultative) bacteria,which are able to convert CO and water or H₂ and CO₂ into ethanol andacetic acid products. Useful bacteria according to this inventioninclude, without limitation, Acetogenium kivui, Acetobacterium woodii,Acetoanaerobium noterae, Clostridium aceticum, Butyribacteriummethylotrophicum, C. acetobutylicum, C. thermoaceticum, Eubacteriumlimosum, C. ljungdahlii PETC, C. ljungdahlii ERI-2, C. ljungdahlii C-01,C. ljungdahlii O-52, and Peptostreptococcus productus. Other acetogenicanaerobic bacteria are selected for use in these methods by one of skillin the art.

By the term “mixed strains,” it is meant a mixed culture of two or moreof the subject bacteria. Such “mixed strains” of the bacteria enumeratedhereinabove are utilized in the methods of this invention.

The terms “bioreactor,” “reactor,” or “fermentation bioreactor,” includea fermentation device consisting of one or more vessels and/or towers orpiping arrangement, which includes the Continuous Stirred Tank Reactor(CSTR), Immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR),Bubble Column, Gas lift Fermenter, Static Mixer, or other devicesuitable for gas-liquid contact. Preferably for the method of thisinvention, the fermentation bioreactor comprises a growth reactor whichfeeds the fermentation broth to a second fermentation bioreactor, inwhich most of the product, ethanol, is produced.

“Nutrient medium” is used generally to describe conventional bacterialgrowth media which contain vitamins and minerals sufficient to permitgrowth of a selected subject bacteria. Sugars are not included in thesemedia. Components of a variety of nutrient media suitable to the use ofthis invention are known and reported in prior publications, includingthose of the inventors. See, e.g. the nutrient media formulae describedin International Patent Publication No. WO98/00558; U.S. Pat. No.5,807,722; U.S. Pat. No. 5,593,886, and U.S. Pat. No. 5,821,111, as wellas in the publications identified above. According to the presentinvention, a typical laboratory nutrient medium for acetate productionfrom CO, CO₂, and H₂ contains 0.9 mg/L calcium pantothenate. However, atypical laboratory nutrient medium for ethanol production from CO, CO₂,and H₂ contains 0.02 mg/L calcium pantothenate.

The terms “limiting substrate” or “limiting nutrient” define a substancein the nutrient medium or gaseous substrate which, during bacterialculture growth in the bioreactor, is depleted by the culture to a levelwhich no longer supports steady state or stable bacterial growth in thebioreactor. All other substances in the nutrient medium or gas substrateare thus present in excess, and are “non-limiting”. The evidence forlimitation is that an increase in the rate of addition of the limitingsubstrate, i.e. in the nutrient feed rate or gas feed rate, to theculture causes a corresponding increase in the rate of gas uptake(mmol/min of gas) due to increase in cell density.

Unless stated otherwise, the term “acetate” is used to describe themixture of molecular or free acetic acid and acetate salt present in thefermentation broth. The ratio of molecular acetic acid to acetate isdependent upon the pH of the system, i.e., at a constant “acetate”concentration, the lower the pH, the higher the molecular acetic acidconcentration relative to acetate salt.

“Cell concentration” in this specification is based on dry weight ofbacteria per liter of sample. Cell concentration is measured directly orby calibration to a correlation with optical density.

The term “natural state” describes any compound, element, or pathwayhaving no additional electrons or protons that are normally present.Conversely, the term “reduction state” describes any compound, element,or pathway having an excess of one or more electrons. The “reductionstate” is achieved by adding one or more electrons to the “naturalstate”, i.e. by lowering the redox potential of the fermentation broth.

“Ethanol productivity” is the volumetric productivity of ethanol,calculated as the ratio of the steady state ethanol concentration andthe liquid retention time (LRT) in continuous systems, or the ratio ofthe ethanol concentration and the time required to produce thatconcentration in batch systems. The phrase “high ethanol productivity”describes a volumetric ethanol productivity of greater than 10 g/L·day.

The phrase “high concentration of ethanol” means greater than about 10g/L, preferably greater than 15 g/L ethanol in fermentation broth or aproduct ratio of ethanol to acetate of 5:1 or more.

“Excess H₂” is available for ethanol production when the ratio of themoles of H₂ in the feed gas to the sum of two times the moles of COconverted and three times the moles of CO₂ converted is greater than1.0. If this ratio is less than 1.0, excess H₂ is not available andethanol can only be produced through a different controlling mechanism.

II. The Biological Pathways Utilized in the Method of this Invention

Without wishing to be bound by theory, the inventors theorize that themethods for increasing the anaerobic production of ethanol from themethods described herein are based upon the biological pathwaysinvolving the conversion of NAD(P)H to NAD(P) in the basic pathwaycycles of the acetogenic pathway for autotrophic growth. The inventioninvolves manipulating those pathways to enable continuous production andmaintenance of high concentrations of ethanol with low acetateconcentrations under stable operating conditions, thereby providingcommercially useful methods for ethanol production from industrialgases.

The essential involvement of NAD(P)H to NAD(P) in the biologicalpathways is described as follows: The production of ethanol from gaseouscomponents, such as CO, CO₂, and H₂ occurs in a three step biologicalmethod. In the first step, the substrates CO and H₂ are oxidized and, indoing so, release NAD(P)H:NAD(P)→NAD(P)HCO+H₂+H₂O→CO₂+4H⁺

The products of step 1 are then converted to acetic acid, a step thatrequires NAD(P)H:NAD(P)H→NAD(P)CO+CO₂+6H⁺→CH₃COOH+H₂OFinally, if excess NAD(P)H is available because the reaction of step 1proceeds at a faster rate than the reaction of step 2, acetic acid isreduced to ethanol.NAD(P)H→NAD(P)CH₃COOH+4H⁺→C₂H₅OH+H₂OThus, the availability of excess NAD(P)H from substrate oxidation leadsto the production of ethanol from acetic acid.

There are two known basic pathway cycles in the acetogenic pathway: (1)the Acetyl-CoA cycle and (2) the THF cycle, in which CO₂ is reduced to amethyl group. The sequence for the generation of ethanol and acetic acidtherefrom is illustrated in J. R. Phillips et al., 1994 AppliedBiochemistry and Biotechnology, 45/46:145. The Acetyl-CoA cycle has aninner cycle, referred to herein as the CO cycle. As the CO cyclenormally reacts clockwise, ferredoxin is reduced. Ferredoxin can also bereduced by H₂ as it is oxidized on the enzyme hydrogenase. As a result,the Acetyl-CoA cycle also reacts clockwise, and ferredoxin is oxidized.If the inner CO cycle and the Acetyl-CoA cycle react at the same rates,ferredoxin is in a redox-state equilibrium. If however, these two cyclesdo not occur at the same rate, i.e., the CO cycle reacts at a fasterrate than the Acetyl-CoA cycle, reduced ferredoxin is built up. Alsowith excess H₂, reduced ferredoxin can also be produced in excess. Thisexcess reduced ferredoxin causes the NAD(P) to be regenerated (reduced)to NAD(P)H, which builds an excess that must be relieved to equilibriumand in doing so, reduces acetic acid to ethanol.

The THF cycle functions for cell growth and is necessary for acontinuous culture; therefore it cannot be completely stopped. Reducingthe THF cycle rate also serves to cause a higher NAD(P)H to NAD(P)ratio. NAD(P)H is oxidized in two places. By limiting this oxidation,which would keep the total cellular NAD(P)H to NAD(P) ratio in balance,the NAD(P)H is used to reduce acetic acid to ethanol.

A second basic method of causing acetic acid to be reduced to ethanol isby directly lowering the redox potential of the fermentation broth. Areduction state sufficiently lower than the natural state of the culturecauses NAD(P)H to be in abundance and promote the reduction of aceticacid to ethanol.

III. The Methods of the Invention

The basic steps of the method include the following: A continuousfermentation method with product recovery is described by reference toFIG. 1 and exemplified in Example 1 below. A continuous flow of gaseoussubstrate 1 comprising at least one reducing gas, e.g., CO or H₂, issupplied at a selected gas feed rate and a continuous flow of liquidphase nutrient medium 2 at a selected nutrient feed rate are supplied toa fermentation bioreactor 3 containing a subject bacteria. In thebioreactor 3, the medium and gaseous substrate are fermented by thebacteria to produce ethanol and acetate acid. Once a stable cellconcentration is achieved under steady state conditions, the componentsof the continuous system are manipulated to reduce the redox potential,or increase the NAD(P)H to NAD(P) ratio, in the fermentation broth,while keeping the free acetic acid concentration in the bioreactor lessthan 5 g/L. The methods of this invention are designed to permit andmaintain production of ethanol and acetate in the fermentation brothsuch that the ethanol productivity is greater than 10 g/L·day at anethanol to acetate ratio of between 1:1 and 20:1. In one embodiment,that ratio is greater than 3:1. In another embodiment, that ratio isgreater than 5:1. In still another embodiment, that ratio is greaterthan 10:1. In still another embodiment that ratio is greater than 15:1.The method of this invention is alternatively effective in enhancingstable continuous (steady state) production of high ethanolconcentrations (15-35 g/L ethanol) and low acetate concentrations (0-5g/L acetate), i.e., ethanol to acetate product ratio of 3:1 or more,from CO, CO₂, and H₂ with good method stability.

Periodically, during the course of the methods of this invention,samples of the broth are removed to determine the ratio by aconventional assay method. For example, the cells are separated from thesample, e.g., by centrifugation and the cell-free sample is then subjectto an assay method, such as the preferred method of gas chromatography.However, other conventional assay methods are selected by one of skillin the art. The additional optional steps of the method are added toachieve and/or maintain the ratio. Example 2 demonstrates such an assaymethod.

Steps used to manipulate the system components and maintain and/orachieve the desired ethanol productivity or the ethanol to acetate ratioinclude at least one, and desirably, combinations of the followingsteps: altering nutrient medium contents, nutrient feed rate, aqueousfeed rate, operating pressure, operating pH, gaseous substrate contents,gas feed rate, fermentation broth agitation rate, avoiding productinhibition step, decreasing cell density in the bioreactor, orpreventing substrate inhibition. Some preferred manipulations includesupplying the bioreactor with liquid phase nutrient (pantothenate orcobalt) limitation, a slight excess of CO and H₂ in the feed gas,minimizing acetate concentration, avoiding culture acclimation to lowliquid phase nutrient concentrations, bringing the culture to a suitablecell concentration at a relatively fast rate, raising the pH of theculture above 4.5, purging bacterial cells from the bioreactor to a cellconcentration less than the stable steady state concentration thatutilizes all reducing gas or nutrient substrates in the bioreactor andincreasing the aqueous feed rate when the free acetic acid portion ofthe acetate present in the fermentation bioreactor broth exceeds 2 g/L,thereby inhibiting any unwanted increase in the concentration of freeacetic acid. All of these steps are described in detail below.

Exhaust gas 4 containing gases other than CO, CO₂ and H₂ and unconvertedCO, CO₂ and H₂ from the reactor are vented from the reactor and are usedfor their fuel value. If excess H₂ as a controlling mechanism isemployed, the H₂ partial pressure in the outlet gas and ratio of H₂partial pressure to CO₂ partial pressure in the exit gas are used toidentify the control of the ethanol to acetate ratio by that step. Cellrecycle is used (but is not required) to increase the concentration ofcells inside the bioreactor, and thus provide more biocatalyst for CO,CO₂ and H₂ conversion. With cell recycle, liquid effluent from thereactor 5 is sent to a cell separator 6 where the cells 7 and permeate(cell free liquid) 8 are separated. The cells 7 are sent back to thebioreactor and the permeate 8 is sent to product recovery.

Cell separation is accomplished by using a continuous centrifuge, hollowfiber or spiral wound filtration system, ceramic filter system or othersolid/liquid separator. Ethanol can be recovered from the permeate (oralternatively the effluent from the reactor 5 if cell separation is notemployed) by a variety of techniques including distillation andadsorption. Permeate 8 is separated in a distillation column to produce95% ethanol overhead 10, and water 11 for recycle back to the reactor 3.The recycle water 11 contains excess nutrients not used in thefermentation, but any excess vitamins from fermentation or cell lysisare destroyed by thermal distillation. The 95% ethanol overhead 10 issent to a molecular sieve 12 where anhydrous ethanol 13, the desiredfinal product, is separated from dilute ethanol 14 which is sent back tothe distillation column 9.

The continuous combination of growth, death and cell purge maintains aconstant cell concentration, such that a continuous method used inproducing ethanol (and small amounts of acetic acid) can operate formany months by being fed CO, CO₂ and H₂ along with nutrients withoutadditional culture supplementation. The methods of this inventionmaintain and control conditions for continuous ethanol and acetic acidproduction and prevent or correct rapidly for method upsets. The methodsof this invention also help prevent culture acclimation to low nutrientconcentration, which can be detrimental to culture performance. In thedescriptions below and in the examples, unless otherwise indicated, thepressure used is 1 atmosphere and the temperature used is between 36-41°C. Desirable temperatures and pressures may be determined by one ofskill in the art, depending on the microorganism selected for use in thebioreactor.

A variety of manipulations, described specifically below, added to thebasic steps of this invention permit the enhanced production of ethanol.Preferably, liquid phase nutrient limitation (pantothenate or cobalt) orthe use of excess H₂ or CO are the method steps of the invention,described in detail below, used to achieve and maintain the desiredethanol productivity and permit production of stable concentrations andratios of ethanol to acetate in the fermentation broth. These conditionspermit production of stable concentrations of ethanol and acetate in thefermentation broth. In a preferred embodiment, the ethanol to acetateproduct ratio produced in the fermentation broth is greater than 10:1and the ethanol concentration is greater than 15 g/L.

A. Calcium Pantothenate Limitation

In one specific embodiment of this invention, the method formanipulating the biological pathways to favor ethanol production andlimit acetic acid production involves limiting the amount of calciumpantothenate in the nutrient medium to an amount which is less thanrequired to maintain the bacteria at a stable, steady stateconcentration that would fully utilize the calcium pantothenateprovided. Pantothenate is a component of Acetyl-CoA and therefore, bylimiting calcium pantothenate in the nutrient medium, the Acetyl-CoAcycle rate is reduced relative to the CO cycle rate. This causes abuild-up of reduced ferredoxin and the reduction of NAD(P) to NAD(P)H,and thereby increases the production of ethanol as the final product.

Pantothenate limitation is observed when the micrograms (μg) of calciumpantothenate fed to the reactor per gram (g) of cells (dry weight)produced in the reactor is in the range of 0.5 to 100. A more desirablepantothenate limitation is in the range of 2 to 75 μg of calciumpantothenate per gram (g) of dry cells produced in the reactor. Still apreferred pantothenate limitation is in the range of 0.5 to 50 μg ofcalcium pantothenate per gram (g) of cells produced in the reactor.Another embodiment of this limitation is at about 1-25 μg of calciumpantothenate per gram (g) of cells produced in the reactor. Anotherembodiment of this limitation is at about 10-30 μg of calciumpantothenate per gram (g) of cells produced in the reactor. This amountof the nutrient maintains ethanol production in preference to acetateproduction. One embodiment of this method is illustrated in Example 4.

In another aspect of this method, the acclimation of the bacteria in thefermentation bioreactor to low limiting calcium pantothenateconcentration is avoided by regulating or adjusting the fermentationparameters, so that a constant calcium pantothenate concentration ismaintained, while at least one, and sometimes more than one, parameterof gas feed rate, liquid feed rate, agitation rate, or H₂ partialpressure is adjusted. Major changes in nutrients are avoided, but arelatively constant nutrient feed concentration is maintained. If theculture is allowed to acclimate to low liquid phase limiting nutrients,poor product ratios of 1.0 g ethanol/g acetate or less occurs in anirreversible method. Thus, reactor shut down and reinoculation isnecessary. Preferably, the biological pathway is controlled to favorethanol production and limit acetic acid production by first supplyingexcess H₂ in the feed gas to the bioreactor, and then limiting calciumpantothenate in the nutrient medium as described above.

In fact, at start-up, the normally limiting liquid phase nutrientcalcium pantothenate is kept in excess to avoid acclimation to lownutrient concentrations, a condition that can result in very poorperformance and the loss of the culture=s ability to produce achievehigh ethanol productivities of more than 10 g/L·day if excess H₂ is notemployed. An example of such regulation of fermentation parameters for aparticular bacterial culture is illustrated in Example 17.

B. Cobalt Limitation

In another embodiment of this invention, the method for manipulating thebiological pathways to favor ethanol production and limit acetic acidproduction involves limiting the amount of cobalt in the nutrient mediumto an amount which is less than required to maintain the bacteria at astable steady state concentration that would fully utilize the cobaltprovided. Cobalt limitation is observed when the micrograms (μg) ofcobalt fed to the reactor per gram (g) of cells (dry weight) produced inthe bioreactor is in the range of 5 to 100. Preferably, a cobaltlimitation involves providing between about 20 to 50 μg of cobalt to thereactor per gram of cells produced in the reactor. This amount of cobaltmaintains ethanol production in preference to acetate in the process.Example 18 illustrates an embodiment of the method of limiting cobalt tothe reactor according to this method.

Limiting cobalt in the fermentation broth may also reduce the Acetyl-CoAcycle rate. Because cobalt is used to transfer a methyl group from theTHF cycle to the Acetyl-CoA cycle, limiting the amount of cobalt in thefermentation broth also reduces the THF cycle function by not permittingthe transfer. Cobalt limitation reduces the THF cycle rate, which alsocauses a higher NAD(P)H to NAD(P) ratio, thereby producing ethanol.

The method is further manipulated by preventing acclimation to lowlimiting cobalt concentration. In much the same manner as acclimation tolow pantothenate concentrations is avoided, a constant cobaltconcentration is maintained while adjusting one or more of thefermentation parameters (gas rate, liquid rate, agitation rate, CO₂content, and H₂ gas partial pressure). Major changes in nutrients areavoided, but instead a relatively constant nutrient feed concentrationis maintained. An example of such regulation of fermentation parametersfor a particular bacterial culture is illustrated in Example 19.

Preferably, the biological pathway is controlled to favor ethanolproduction and limit acetic acid production by first feeding excess H₂to the reactor and then limiting cobalt in the nutrient medium asdescribed above. At start-up, the limiting liquid phase nutrient cobaltis kept in excess to avoid acclimation to low nutrients concentration, acondition that can result in very poor culture performance and the lossof the cultures ability to produce product ratios greater than 1:1.

C. Oversupplying Hydrogen

In still another embodiment, the method for manipulating the biologicalpathways to favor ethanol production and limit acetic acid productioninvolves feeding excess H₂ in the feed gas or limiting gaseous carbonwhich results in excess H₂, which is then used by the biologicalpathway. Preferably, the H₂ reducing gas is in excess relative to CO,and the excess H₂ causes the bacteria to produce a high ethanol toacetate ratio in the fermentation broth. If the ratio of the H₂ (molesof gas fed) to the sum of two times the CO (in moles of gas) convertedand three times the CO₂ (in moles of gas) converted is greater than 1,the fermenter is carbon limited. The H₂ partial present in the exit gasis preferably greater than 0.4 atm. Finally the ratio of H₂ partialpressure to CO₂ partial pressure must be greater than 3.0 to assure thatsufficient H₂ is available to use all the CO₂. If the CO₂ partialpressure is greater than 0.1 atm, it is likely that growth has beenotherwise limited. See, Example 20 for an illustration of this methodstep.

During start-up, the use of excess H₂ is favored over nutrientlimitation, mainly because it is easier to control. The benefits ofemploying excess H₂ are that it avoids excess acetic acid production,which can lead to poor product ratios and potential acetic acidinhibition, as well as acclimation to low nutrient concentrations.

D. Oversupplying Carbon Monoxide

Another way of manipulating the components of the method involvesoversupplying the reducing gas, CO, in the gaseous substrate for use inthe pathway, which serves to directly lower the redox potential in thefermentation broth. Thus, according to this embodiment, the bioreactoris supplied with gaseous substrate comprising CO where the amount of COpresent in the bioreactor is greater than the amount required tomaintain the bacteria at a stable, steady state concentration that wouldfully utilize the CO provided. CO oversupply as a method of favoringethanol production over acetic acid production when the specific rate ofCO uptake (millimoles of CO per gram of cells (dry weight) in thereactor per minute, or mmol/g cell·min) is greater than 0.3. Morepreferably, this step involves a specific rate of CO uptake of greaterthan 0.5. This means that each cell on the average is utilizing CO inits metabolism at a rate of at least 0.3 mmol/g·min., or more ideally ata rate of at least 0.5 mmol/g·min. Preferably, the CO is provided at arate at which the CO uptake is from 0.3 to 2 mmol CO/gram cell (dryweight) of bacteria/minute. In another embodiment, the CO is provided ata rate of from 0.5 to 1.5 mmol CO/gram cell (dry weight) ofbacteria/minute. In another embodiment, the CO is provided at a rate ofabout 1 mmol CO/gram cell (dry weight) of bacteria/minute. Example 24provides an illustration of one embodiment of this method step.

This rate of CO uptake maintains ethanol production in preference toacetate production. If CO is supplied such that the dissolved CO in thefermentation broth is significant by gas pressure or extremely good masstransfer, the fermentation broth becomes more reduced. Oversupply of COhas two additional benefits. Excess CO may cause the CO cycle to operateat a faster rate, and if the Acetyl-CoA cycle is otherwise limited andcannot keep up with the CO cycle, reduced ferredoxin builds-up. CO mayalso slow down step 2 (production of the intermediate acetic acid) inthe overall three-step method through substrate inhibition. Thisdecreased rate of step 2 in relation to step 1 causes an excess ofNAD(P)H, which leads to ethanol production in favor of acetic acid.

Although excess CO can result in increased ethanol production bydirectly reducing the redox potential of the fermentation broth, thepresence of excess CO also inhibits growth by inhibiting theCO-dehydrogenase and therefore the uptake of H₂. The presence of excessCO unfortunately also results in poor H₂ conversion, which may not beeconomically favorable. The consequence of extended operation undersubstrate inhibition is poor H₂ uptake. This eventually causes celllysis and necessary restarting of the reactor. Where this method has anunintended result of CO substrate inhibition (the presence of too muchCO for the available cells) during the initial growth of the culture orthereafter, the gas feed rate and/or agitation rate is reduced until thesubstrate inhibition is relieved. An illustration of how to adjust thegas rate or agitation rate to accomplish this effect is illustrated inExample 21.

E. Additional Manipulating Steps

In addition to the major method enhancing steps described above, severalmethod steps are desirably included in the ethanol production method.

1. Increasing Mass Transfer

One such additional embodiment involves ensuring that the mass transferof the CO or H₂ from the gas feed to the liquid fermentation broth isfaster than the ability of the bacteria to utilize the dissolved gases.For example, if a bioreactor containing C. ljungdahlii is fed CO, CO₂and H₂ and is operated without limitation on nutrients (such aspantothenate or cobalt) or the presence of excess H₂, cell growth islimited by the amount of gas transferred into the liquid phase and thesystem produces acetic acid as the product. If the culture is fed aslight amount of CO or H₂ in excess of that required for culture growth,it produces ethanol. However, if too much gas is transferred into theliquid phase for the culture to use, substrate inhibition occurs, whichcan lead to culture upset and cell death. Thus, there is a very narrowrange of operation with excess mass transfer. Example 22 provides anillustration of this method.

With reference to the Acetyl-CoA cycle, in order for the excess reducedferredoxin to be produced, the CO cycle or the reduction of ferredoxinthrough hydrogenase must occur faster than the Acetyl-CoA cycle. Themethods described herein limit the rate at which the organisms canutilize the dissolved gases by restricting the rate at which essentialnutrients e.g., calcium pantothenate or cobalt, or other substrates,such as CO₂ gas, are available to the bacteria, or by providing excesssubstrate, H₂ or CO to the culture.

A theoretical rate of mass transfer, which is faster than the rate atwhich the bacteria can use substrate, even without other limitations,can be calculated. That rate, when achieved, is limited by the naturalgrowth rate of the organism. Therefore, the most productive embodimentis where the mass transfer (gas flow rate or agitation rate) is fasterthan the rate at which the highest possible concentration of cells canutilize the substrate without any limitation. There would be a verynarrow operating range since substrate inhibition could quickly causecell death and a resulting by-product concentration which is toxic tothe culture.

2. Supplying Excess CO and H₂

In another embodiment of a method of this invention, stability in thehigh ethanol concentration/limited acetic acid production is achieved inthe methods which limit cobalt or calcium pantothenate, or provide anabundance of H₂ or CO. According to this step, as the culture uses thegaseous substrates CO, H₂ and CO₂ as the carbon and energy sources, COand H₂ are supplied in slight excess. A slight excess of CO and H₂ isachieved by attaining steady operation and then gradually increasing thegas feed rate and/or agitation rate (10% or less increments) until theCO and H₂ conversions just start to decline. This is one means ofavoiding mass transfer limitation, which favors acetic acid production,and supplying excess reduced ferredoxin in order to reduce NAD(P) toNAD(P)H and produce ethanol. If CO and H₂ are not supplied in slightexcess, mass transfer limitation occurs, and the pathway is balanced.This results in poor ethanol to acetate product ratios (high acetateconcentrations). High acetate concentrations can ultimately result inacetic acid inhibition, which limits the ability of the bacterium totake up H₂ and can eventually lead to culture failure.

Steps to avoid mass transfer limitation include an increase in theagitation rate or gas rate to transfer more CO and H₂ into the liquidphase, and thus return to the presence of a slight excess CO and H₂. Ifproduct inhibition occurs as a result of mass transfer limitation, it isnecessary to increase the liquid feed rate to clear the acetic acidinhibition, by diluting to a lower resulting acetate concentration.Since increasing the medium feed rate would increase the μg pantothenateor cobalt/g-cell produced, this must be done only briefly or the excesspantothenate or cobalt must be eliminated by adjusting the mediumconcentration or increasing the water feed rate.

3. Controlling Acetic Acid Product Inhibition

Where in the methods described above, acetic acid product inhibition canoccur if too much molecular acetic acid, i.e., >2 g/L, accumulates inthe bioreactor to allow cell growth and further ethanol production.Another manipulating step is used to avoid culture failure. Onemodification involves briefly increasing the liquid or aqueous feed rateto reduce the liquid phase concentration of inhibiting acetic acid tolower than 2 g/L. An illustration of this step for a particular culturein a reactor is demonstrated in Example 23.

4. Water Recycle Step

Still another optional method step for maintaining a stable culturewhich produces ethanol as the only product with no net acetic acidproduction in the methods of this invention involves adding waterrecycle from distillation back to the fermentation reactor (see, e.g.,Example 15). As was noted earlier, water (containing up to 5 g/Lacetate) recycle has the benefit of recycling the produced acetate backto the reactor so that no net acetic acid is produced. An equilibrium isthus established between the ethanol and acetate in the reactor. As aresult, all CO, CO₂ and H₂ fed to the reactor and converted to productsresults in ethanol production, except for that used for culturemaintenance.

5. Reducing Cell Density

Still another manipulating step useful in the method is to initiateperiodic or continuous purging of bacterial cells from the bioreactor toreduce the cell concentration in the bioreactor. This manipulationserves to reduce the cell concentration to less than a stable, steadystate cell concentration that utilizes all reducing gas or nutrientsubstrates in the bioreactor. By thus, altering the cell density, theproduction of ethanol is favored over the production of acetate in thebioreactor. See, e.g., Example 25.

6. Two Stage CSTR

One of the problems associated with ethanol production with mediumlimitation is the ability or tendency of the culture to eventually adaptto the limiting conditions and not continue to produce ethanol afterseveral months of operation. Instead acetate eventually becomes thedominant product. This acclimation to low limiting nutrientconcentrations results in a culture which produces more acetic acid thanethanol (ethanol to acetate product ratio of 1.0 or less), and yieldslow ethanol concentrations (sometimes as low as 1 g/L). Adaptation mostlikely occurs when the culture is not provided with sufficient nutrientsduring start-up, where growth rate is more important than ethanolproduction rate. Additionally, there is a danger that the culture can beacclimated to low limiting nutrient concentrations during steady stateoperation particularly as the limiting nutrient concentrations areadjusted downward to rid the reaction system of acetate.

To avoid this adaptation when using the pantothenate or cobalt limitingsteps above, instead of allowing the culture to grow with the availablenutrients, and the danger mentioned above, another modification of themethod can be employed. A two-stage CSTR system where primarily goodculture growth occurs in the first stage on a slight excess of limitingnutrients (perhaps with accompanying acetic acid production), followedby a production stage where the culture from the first stage is nowlimited by the limiting nutrient and is used to produce highconcentrations of ethanol, is another modification of the method. Thismodification enables the maintenance of a stable culture, which does notacclimate to reduced pantothenate or cobalt concentrations. Thismodification involves operating a two-stage CSTR, in which a growthreactor (Stage 1) to feed a production reactor (Stage 2) where the bulkof the ethanol production occurs. The growth reactor is not operatedwith the nutrient limitation steps described above, so the culture isnot as susceptible to acclimation to a limited condition.

A schematic diagram of this two-stage CSTR system is shown in FIG. 2,and the following description has reference to that figure. According tothis embodiment, the Growth Stage is operated at a liquid retention time(LRT) of about 24 hours. The Growth Stage CSTR 1 is fed enoughpantothenate or cobalt in the medium 2 to yield a healthy culture (andmay produce some acetic acid as well). Thus, excess acetic acid isproduced in the reactor, but with increased stability. This pantothenateor cobalt concentration is in excess of what would normally be fed to asingle CSTR used to produce ethanol. The gas feed to this reactor isunconverted gas 3 from the Production Stage 4 and the liquid feed isfresh medium 2. The Growth Stage CSTR is operated without cell recycle.The purpose of this Growth Stage reactor is to provide a healthy culturefor later ethanol production that does not acclimate to low pantothenateconcentrations.

The Production stage reactor 4 is operated at a nominal LRT of less than20 hours. This CSTR with cell recycle is fed a fresh gas feed 5, and mayhave low conversions. It is fed fresh medium feed 6 as well as culturefeed 7 from the Growth Stage. Minimal pantothenate or cobalt is fed tothis reactor since the excess from the Growth Stage is available. Cellrecycle 8 is used in this reactor in order to get the most productionout of the cells sent back to the reactor 9. The exit ethanolconcentration in the liquid product 10 should be greater than 20 g/L.The features of the two-stage CSTR system include little change foracclimation to low pantothenate or cobalt concentrations; an overall LRTof less than or equal to 30 hours; an expected greater ethanolproductivity and higher ethanol concentration than from a single CSTR ofthe same size.

7. Start-Up Modifications

Still other method steps, which are preferably utilized in the practiceof this invention, involve cell production in the initial start-up ofthe fermentation culture. The start-up of a bioreactor fed CO, CO₂ andH₂ to produce ethanol and acetic acid is accomplished by batchinoculation from stock culture (Example 11) or by employing a continuousinoculum from an existing reactor as culture feed (Example 12). As notedearlier in the discussion of avoiding culture acclimation to lowpantothenate or cobalt concentrations, the culture is most desirablybrought up to a high cell concentration prior to limiting nutrients, butsupplying excess H₂ to the culture. This rapid start-up avoids cultureacclimation and yields good product ratios (high ethanol and low acetateconcentrations). If the rapid start-up is not employed, poor productratios can occur and the culture can acclimate to low liquid phasenutrient concentrations and require reactor reinoculation.

The reactor is started with a batch liquid phase (liquid medium is notinitially fed continuously to the reactor), at low agitation rates(perhaps 400-600 rpm in a laboratory New Brunswick Scientific Bioflo®reactor) and at the desired pH. The liquid phase in the reactor thusconsists of a batch of nutrient medium containing vitamins and salts,with a nominal concentration of limiting nutrient, either calciumpantothenate or cobalt (20 μg/L pantothenate or 75 ppb cobalt as anexample). If continuous inoculum from an existing reactor is employed,batch liquid phase operation likely is not necessary. In this case, gasis fed continuously to the reactor during initial start-up at a slowrate. Ideally, the gas phase at start-up would be CO₂-free, H₂-abundantand the gas rate and agitation rate would be kept at low levels to avoidCO substrate inhibition.

An exemplary general start-up protocol for producing and sustainingcommercially viable ethanol concentrations from CO, CO₂ and H₂ consistsof three distinct phases: (a) initial start-up, where cell production iscritical; (b) start-up where production rate becomes critical; and (c)steady state operation. Essentially, initial start-up is characterizedby inoculation of a batch liquid, with a nominal limiting nutrient,selected from cobalt (75 ppb) or calcium pantothenate (20 μg/L) at adesired pH (typically 4.5-5.5). To facilitate start-up, the gas feedrate and agitation rate are preferentially kept low, while H₂ is fed inexcess. The cause of ethanol production during start-up is excess H₂;nutrient limitation occurs later. Thus, excess liquid nutrients areactually present during start-up to avoid unwanted culture acclimationto low nutrients. As the fermentation proceeds over a period of severalhours after inoculation, CO₂ is produced and H₂ is consumed. The changesin these rates indicated that the agitation rate should be nominallyincreased slowly (perhaps by 200-300 rpm in a laboratory reactor, over aperiod of 2-3 days) to avoid mass transfer limitation.

This onset of CO₂ production occurs much more rapidly in systemsemploying continuous inoculation as opposed to batch inoculation fromstock culture. However, if the agitation rate is increased too fast, COsubstrate inhibition occurs. This procedure of watching H₂ conversion(or CO₂ production) while nominally increasing agitation rate occurs ata relatively rapid rate until the target agitation rate is reached.During this time of increasing agitation rate in batch liquid culture,cell production instead of product formation is of utmost importance.

Once the target agitation rate is reached (800-1000 rpm in laboratoryNew Brunswick Scientific Bioflo® reactor), the culture is allowed tosteady to confirm H₂ uptake. The start-up shifts to a mode in whichproduction rate becomes important. It is desirable to have COconversions exceeding 80% and a high H₂ partial pressure in the exit gas(at least 0.55 atm) to assure ethanol production while limiting acetateand the free molecular acetic acid concentration. The liquid medium feedrate is then turned on (for systems having batch inoculation from stockculture) to initiate continuous liquid feed and the gas rate isincreased in 10% increments toward the target flow rate. H₂ remains inexcess to avoid excess acetic acid production. As the gas rate isincreased, the liquid phase nutrients are limited (calcium pantothenateor cobalt), and the effect of such limitation is a small drop in H₂conversion, at the target production.

At steady state operation, production of 15-35 g/L ethanol and 0-5 g/Lacetate is reached. At this stage, small adjustments in limitingnutrients, liquid feed rates and gas feed rates are needed, and arechosen by one of skill in the art with resort to knowledge extant in theart as well as the teachings of this invention. If cell recycle is to beadded to the method of ethanol production, it is added at this timealong with an adjustment in gas rate (increase) and nutrientconcentration (decrease).

The above described methods of continuously producing and maintaininghigh concentrations of ethanol with low by-product acetateconcentrations under stable operating conditions enhance the use of thesubject bacteria on a commercial scale for ethanol production. The stepsoutlined in the methods above overcome the limitations of utilizing thesubject bacteria for commercial ethanol production from CO, CO₂ and H₂.Preferably the method employs a continuous bioreactor, although batchand fed-batch fermentation methods are also used, but are not likely tobe economically viable for large-scale ethanol production.

The following examples illustrate various aspects, methods and methodsteps according to this invention. These examples do not limit theinvention, the scope of which is embodied in the appended claims.

Example 1 An Exemplary Method of the Present Invention

A synthesis or waste gas containing CO and/or CO₂/H₂ is continuouslyintroduced into a stirred tank bioreactor containing a strain of C.ljungdahlii, along with a conventional liquid medium containingvitamins, trace metals and salts. One desirable nutrient medium isreported in Table 1 below.

During method start-up using a culture inoculum of 10% or less thereactor is operated with a batch liquid phase, where the liquid mediumis not fed continuously to the reactor. The liquid phase in the reactorthus consists of a batch of nutrient medium with a nominal concentrationof limiting nutrient, either calcium pantothenate or cobalt.Alternatively, a rich medium containing yeast extract, trypticase orother complex nutrients can also be employed.

Ideally, the gas phase at start-up is CO₂-free and contains excess H₂.The gas rate and agitation rate are kept at low levels (less than 500rpm in a New Brunswick Scientific Bioflo® fermentation bioreactor) toyield CO and H₂ in slight excess, but at the same time, avoiding COsubstrate inhibition. In a one-liter laboratory New Brunswick ScientificBioflo® fermentation bioreactor, as an example, where the feed gascomposition is 63% H₂, 32% CO and 5% CH₄, the agitation rate to initiatestart-up is 400 rpm and the gas rate is 20 mL/min. The cause of ethanolproduction during start-up is excess H₂; limitation on nutrients occurslater. Thus, excess liquid nutrients (pantothenate, cobalt) are actuallypresent during start-up to avoid unwanted culture acclimation to lownutrients.

As the fermentation proceeds over a period of several hours afterinoculation, CO₂ is produced from the conversion of CO, and H₂ isconsumed along with the CO₂, which is a signal to nominally increase theagitation rate to avoid gas mass transfer limitation. In the NewBrunswick Scientific Bioflo® CSTR, the exit gas is 25% CO, 67% H₂, 2%CO₂, and 6% CH₄. If the agitation rate is increased too fast, COsubstrate inhibition occurs, as evidenced by a decrease in methaneconcentration after an increase in agitation. Thus the agitation ratemight typically be increased by 200 rpm in 24 hours. This procedure ofmonitoring CO₂ production (or H₂ conversion) while nominally increasingagitation rate occurs at a relatively rapid rate until the targetagitation rate is reached. A typical target agitation rate in the NewBrunswick Scientific Bioflo® fermentation bioreactor is 900 rpm. Duringthis time of increasing agitation rate in batch liquid culture, cellproduction instead of product formation is of utmost importance. Thus,cell concentrations of about 1.5 g/L are attained, while typical productconcentrations are 10 g/L ethanol and 2 g/L acetate from the batchculture.

Once the target agitation rate is reached, the system is allowed to growto maximum H₂ uptake. It is desirable to have very high H₂ exitconcentrations (typically >60%) to assure ethanol production whilelimiting acetic acid production. The liquid medium feed is then turnedon (for systems having batch inoculation from stock culture) to initiatecontinuous liquid feed and the gas feed rate is increased toward thetarget flow rate. In the laboratory New Brunswick Scientific Bioflo®fermentation bioreactor the liquid feed rate is typically 0.5 mL/min,while the gas flow rate is increased by 10 to 15% every 24 hours towarda target rate of 125 mL/min.

It is important to provide excess H₂ in the feed gas to avoid excessacetic acid production. As the gas flow rate is increased, cellproduction increases until the reactor is eventually limited on liquidphase nutrients (calcium pantothenate or cobalt) as evidenced by a smalldrop in H₂ conversion, at the target productivity. In the New BrunswickScientific Bioflo® CSTR, this is recognized by a 10% drop in H₂conversion at a target productivity of 20 g/L·day.

The production method and reactor system are then maintained at a steadystate producing 15 to 35 g/L ethanol and 0 to 5 g/L acetate as products,with only occasional small adjustments in limiting nutrients, liquidrates and gas rate. Typical steady state conditions in the laboratoryNew Brunswick Scientific Bioflo® fermentation bioreactor without cellrecycle, are a gas retention time (gas flow rate/reactor liquid volume)of 20 minutes, a liquid retention time (liquid flow rate/reactor liquidvolume) of 30 hours and an agitation rate of 900 rpm, yielding COconversions of 92% and H₂ conversions of 60% with pantothenatelimitation.

In an embodiment of this method in which cell recycle is added to thereactor system, it is added at this time along with an adjustment in gasrate (increase) and nutrient concentration (decrease). With cell recyclein the New Brunswick Scientific Bioflo® CSTR, the gas retention time istypically 8 minutes, the liquid retention time is 12 hours, the cellretention time is 40 hours and the agitation rate is 900 rpm. Theseconditions typically yield a CO conversion of 92% and a H₂ conversion of50% with pantothenate limitation.

Example 2 Sample Analysis Via Gas Chromatography

To achieve and/or maintain proper productivity and ratio, a sample ofthe fermentation broth in the fermentation bioreactor must beperiodically sampled. A sample greater than 1.5 mL of culture is takenfrom the culture in the bioreactor. The sample is placed in amicrocentrifuge tube and the tube is placed in a Fisher Scientific Micro14 centrifuge with necessary ballast for balancing. The sample issubjected to 8000 rpm for 1.5 minutes. A 0.500 mL sample of supernatantis placed into a 1.5 mL vial designed for use in a gas chromatographautosampler. A 0.500 mL sample of an internal standard solutioncontaining 5 g/L of n-propanol and 5% v/v, 85% phosphoric acid indeionized water. The phosphoric acid assures the all acetate isconverted to acetic acid and is detected by gas chromatography.

One μl of the prepared sample is then injected by autosampler into aHewlett-Packard 5890 Series II Gas Chromatograph equipped with a 007 FFAQuadrex 25 m×0.53 mm ID fused silica capillary column. The analysis isconducted with a helium carrier gas in split-flow mode with 66 mL/minsplit-flow and 7.93 mL/min injector purge. The column head pressure isset to 4 psig which yields a column carrier flow of 7 mL/min. Thetemperature program is 75° C. for 0.2 minutes, a ramp to 190° C. at arate of 30° C./minute, and a hold time at 190° C. for 5.17 minutes. Theresulting runtime is 8 minutes. The instrument is calibrated for ethanol(0 B 25 g/L), acetic acid (0-25 g/L), n-butanol (0-5 g/L) and butyricacid (0-5 g/L). Five standards, prepared from reagent grade materials,are used for the calibration. If the sample is outside the calibrationrange of concentration (e.g., >25 g/L ethanol), 0.250 mL of the sampleand 0.250 mL of deionized water are placed into the vial with 0.500 mLof the internal standard and the dilution factor is included in theanalysis.

Example 3 Acid Production in a Laboratory CSTR with Cell Recycle

A New Brunswick Scientific Bioflo® laboratory fermentation bioreactorwas operated with cell recycle using Clostridium ljungdahlii, strainERI-2, ATCC 55380 for the production of acetic acid from CO, CO₂ and H₂.The gas feed contained 40% H₂, 50% CO and 10% N₂, and the gas retentiontime to the one-liter reactor was 7.7 to 8.6 minutes. Liquid mediumcontaining vitamins, salts and trace elements was fed at a liquidretention time of 2.6 to 2.9 hours. The pH was 5.1 to 5.2, the agitationrate was 1000 rpm and the cell retention time was about 40 hours. Underthese conditions of mass transfer limitation (and not nutrientlimitation), the CO conversion was 94 to 98% and the H₂ conversion was80 to 97%. The cell concentration was 4 to 8 g/L, and acetate wasproduced at 10 to 13 g/L. No ethanol was produced. Although the reactorwas operated under mass transfer limitation (limited by the ability totransfer gas to the culture) and thus produced only acetic acid as theproduct, the parameters for ethanol production through pantothenatelimitation, cobalt limitation or the presence of excess H₂ or CO weremonitored to serve as comparisons for when ethanol is produced as thedominant product.

As shown in Table 2, the Ca-d-pantothenate fed per unit of cellsproduced was 1575 to 3150 micro-grams per gram of cells produced(μg/g-cell produced). Similarly the cobalt fed per gram of cellsproduced was 1734 to 3468 (μg/g-cell produced). The specific CO uptakerate was 0.35 to 0.61 mmol/g-cell·minute. The ratio of the moles of H₂fed to the sum of two times the moles of CO converted and three timesthe moles of CO₂ converted was less than 0.46. Thus, none of theparameters were in the desired operating range for ethanol production bythe culture.

It is realized that pantothenate and cobalt were fed in large excess tothe reactor above when making acetic acid as the product under masstransfer limitation. That is, the pantothenate and/or cobalt levelscould be decreased significantly and still be above the levels forpantothenate or cobalt limitation. To illustrate this, the medium fed tothe 1-liter New Brunswick Scientific Bioflo® fermentation bioreactor wasmodified to significantly decrease cobalt addition to a level that wasjust above the concentration of cobalt for cobalt limitation. Thereactor again contained C. ljungdahlii strain ERI-2 for production ofacetic acid from CO, CO₂ and H₂. The gas feed contained 55% H₂, 25% CO,15% CO₂ and 5% CH₄ (reference gas), and the gas retention time was 7.5to 8.0 minutes. Liquid medium containing salts, vitamins and traceelements was fed at a liquid retention time of 3.0 to 3.5 hours, and thecell retention time was 40 hours. The pH was 5.0 to 5.3 and theagitation rate was 900 to 1000 rpm. Under these conditions the COconversion was 95 to 99% and the H₂ conversion was 94 to 98%. The cellconcentration was 2.5 to 4.0 g/L and acetate was the only product at 10to 14 g/L.

The Ca-d-pantothenate fed to the reactor per gram of cells was 2250 to3600 μg pantothenate/g-cells produced. The cobalt fed per unit of cellsproduced was reduced to a range of 62.0 to 99.2 μg cobalt/g-cellsproduced. The specific CO uptake rate was 0.325 to 0.4mmol/g-cell·minute. The ratio of H₂ fed to the sum of two times the COconverted and three times the CO₂ converted was 0.875.

Example 4 Ethanol Production in Laboratory CSTRs with PantothenateLimitation

A New Brunswick Scientific Bioflo® II laboratory fermentation bioreactorwas operated as a straight through CSTR (without cell recycle) using C.ljungdahlii, strain C-01 ATCC 55988 for the production of ethanol fromCO, CO₂ and H₂, limited on pantothenate. The gas feed to the reactorcontained 63.3% H₂, 31.4% CO and 5.3% C₂H₆ (reference gas); fed at a gasretention time of 27 minutes. Liquid medium containing excess salts andtrace elements and a limited supply of pantothenate was fed to the 1.55liter reactor at a liquid retention time of 31.4 hours. The pH was 4.6to 4.7, and the agitation rate was 650 rpm. Under these operatingconditions the CO conversion was 98%, the H₂ conversion was 83% and thecell concentration was 1.5 to 2.0 g/L. Ethanol was produced at aconcentration of 15 to 19 g/L, and acetate was produced at 1.5 g/L. Theethanol productivity ranged from 11.5 to 14.5 g/L·day.

In analyzing the parameters for ethanol production, pantothenatelimitation was seen by operating with a pantothenate feed to cellproduction ratio of 17.7 to 23.6 μg pantothenate/g-cell produced.Compare this ratio to the 2250 to 3600 μg pantothenate/g-cell producedand 1575-3150 μg pantothenate/g-cell produced in Example 3 for acidproduction. The cobalt fed per unit of cells produced was 5000 to 6000μg cobalt/g-cell produced, a level that is even greater than in Example3 and assures no cobalt limitation. The specific CO uptake rate was 0.23to 0.30 mmol/g-cell·minute. The ratio of H₂ fed to the sum of two timesthe CO converted and three times the CO₂ converted was 1.03, and the H₂partial pressure in the exit gas was 0.55-0.64 atm. It is possible thateither excess H₂ or limited pantothenate caused ethanol production.

Pantothenate limitation for ethanol production was also addressed inanother New Brunswick Scientific Bioflo® II laboratory reactor operatedwith cell recycle using C. ljungdahlii, strain C-01 ATCC 55988. Thisreactor was fed gas containing 61.7% H₂, 30.6% CO and 5.2% C₂H₆(reference gas) at a gas retention time of 12.3 minutes. Liquid mediumcontaining a limited supply of pantothenate along with excess salts andtrace elements was fed to the 2.4 liter reactor at a liquid retentiontime of 24.8 hours. Cell recycle was provided by employing a 0.2 μmhollow fiber membrane, and the cell retention time was 69 hours. The pHwas 4.6, and the agitation rate was 650 rpm. Under these conditions theCO conversion was 90%, the H₂ conversion was 53% and the cellconcentration was 2.5 g/L. The ethanol concentration was 18 g/L and theacetate concentration was 3 g/L. The ethanol productivity was 17.4g/L·day.

In analyzing the parameters for ethanol production (Table 2), the ratioof pantothenate fed per unit of cells produced was 8.08 μgpantothenate/g-cell produced. Again, pantothenate limitation was assuredby operating at a level far less than that required for acetateproduction. The cobalt fed per unit of cells produced was 3960 μgcobalt/g-cell produced. The specific CO uptake rate was 0.33mmole/g-cell·minute. The ratio of H₂ fed to the sum of two times the COconverted and three times the CO₂ converted was 1.14, and the H₂ partialpressure in the exit gas was 0.60-0.65 atm. Excess H₂ could be apotential reason for ethanol production; however, the high CO₂ contentin the exit gas (0.14 atm) shows that growth was limited bypantothenate.

In another experiment, C. ljungdahlii, strain ERI-2 was fed 1500 to 3600μg pantothenate/g cells produced during acetic acid production from CO,CO₂ and H₂, a condition where the reactor was not limited onpantothenate (or any other limitation except for the ability to transfergas to the culture), and no ethanol was found in the product stream.

During limitation on pantothenate for ethanol production from CO, CO₂and H₂ , C. ljungdahlii, strain C-01 was fed 8 to 24 μg pantothenate/gcells produced, while maintaining all other nutrients in excess. Underthese conditions, strain C-01 produced 15 to 19 g/L ethanol and 1.5 to3.0 g/L acetate.

Example 5 Ethanol Production in Laboratory CSTRs with Cobalt Limitation

A New Brunswick Scientific Bioflo® II laboratory fermentation bioreactorwas operated as a straight through CSTR (with no cell recycle) using C.ljungdahlii, strain C-01, ATCC 55988 for the production of ethanol fromCO, CO₂ and H₂ with cobalt limitation. The gas fed to the reactorcontained 60% H₂, 35% CO and 5% CH₄ (reference gas), and was fed at agas retention time of 14 minutes. Liquid medium containing excess salts,vitamins and trace metals (except for cobalt, which was limiting) wasfed to the 2.5 L reactor at a liquid retention time of 40 hours. The pHwas 4.9 and the agitation rate was 650 rpm. Under these conditions theCO conversion was 91%, while the H₂ conversion varied from 20 to 80%,but was nominally 55%. Ethanol was produced at 26 g/L, acetate wasproduced at 4 g/L and the cell concentration was 2.5 g/L. The ethanolproductivity was 15.6 g/L·day.

In analyzing the parameters for ethanol production, the ratio of thepantothenate fed to the cell production was 15.2 μg pantothenate/g-cellproduced. This level was quite low, such that cobalt limitation mightnot be assured in favor of pantothenate limitation. Cobalt limitationwas seen by operating with 33.3 μg cobalt/g-cell produced, a level whichis 100 times less than used in reactors without cobalt limitation. Theratio of the H₂ fed to the sum of two times the CO converted and threetimes the CO₂ converted was 0.94. The specific CO uptake rate was 0.37mmole/g-cell·minute.

Cobalt limitation for ethanol production was also demonstrated in a CSTRwith cell recycle using C. ljungdahlii, strain C-01 ATCC 55988. Thisexperiment was run to demonstrate cobalt limitation in the presence ofexcess pantothenate, in contrast to the previous reactor in thisexample. The New Brunswick Scientific Bioflo® 2000 laboratoryfermentation bioreactor with a 0.2 μm hollow fiber membrane for cellrecycle, was fed gas containing 60% H₂, 35% CO and 5% CH₄ (referencegas) at a gas retention time of 5 minutes. Liquid medium containingexcess salts, vitamins and trace metals (again, except for cobalt whichis limiting) was fed to the 1.2 liter reactor at a liquid retention timeof 16 hours. The pH was 5.1 and the agitation rate was 825 rpm. The cellretention time in this CSTR with hollow fiber for cell recycle was 40hours. Under these conditions the CO conversion was 83%, the H₂conversion was 50% and the cell concentration was 4.2 g/L. The ethanolconcentration was 18 g/L and the acetate concentration was 4 g/L. Theethanol productivity was 27 g/L·day.

In addressing the parameters for ethanol production in this reactor(Table 2), the ratio of pantothenate fed to cell production was 85.7 μgpantothenate/g-cells produced, a level which is 5.5 times greater thanin the previous reactor in this example. Cobalt limitation was seen byoperating with 47.6 μg cobalt/g-cells produced. The ratio of H₂ fed tothe sum of two times the CO converted and three times the CO₂ convertedwas 1.03, and the H₂ partial pressure in the exit gas was 0.60 atm.Again, excess H₂ could be a potential reason for ethanol production;however, the high CO₂ content in the exit gas (0.1-0.15 atm) shows thatgrowth was limited by cobalt. The specific CO uptake was 0.50mmol/g-cell·minute.

Example 6 Ethanol Production in Laboratory CSTRs when Operating withExcess CO Present

A high pressure AUTOKLAV™ reactor (Buchi) was operated as a CSTR withculture circulation and cell recycle using C. ljungdahlii strain C-01for the production of ethanol from CO, CO₂ and H₂ in the presence ofexcess CO for a period of 50 hours. The reactor was operated at 25 psigand fed gas containing 57% H₂, 36% CO and 6% C₂H₆. The gas retentiontime was variable, but was nominally 3.0 minutes. Liquid mediumcontaining excess salts, vitamins (including pantothenate) and tracemetals was fed to the 600 mL reactor at a liquid retention time of 8.2hours. The cell retention time, obtained by passing the reactor effluentthrough a ceramic hollow fiber filter, was 18.5 hours. The pH was 4.5,the agitation rate was 450 rpm and the liquid recirculation rate was 0.4to 0.5 gpm. Under these conditions, the gas conversions were variable,but the CO conversion was nominally 72% and the H₂ conversion wasnominally 12%. The cell concentration was 2.7 g/L. Ethanol was producedat 9.9 g/L and acetate was produced at 2.6 g/L. The ethanol productivitywas 29.0 g/L·day.

In analyzing the parameters for ethanol production, the ratio of thepantothenate fed to the cell production was 97 μg pantothenate/g-cellproduced. This level is sufficiently high to assure that pantothenatewas not limiting. The ratio of cobalt fed to the cell production was 836μg cobalt/g cell produced, again a level that assures that cobalt wasnot limiting. The ratio of the H₂ fed to the sum of two times the COconverted and three times the CO₂ converted was 1.09, and the H₂ partialpressure was 1.6 atm. The high CO₂ content in the exit gas (0.5 atm)assures that excess H₂ did not cause ethanol production. The specific COuptake rate was 1.34 mmol/g-cell·min., a level that assures excess CO asa method of producing ethanol.

The technique of using excess CO for ethanol production was alsodemonstrated in another experiment with C. ljungdahlii, strain C-01 inthe AUTOKLAV™ reactor (Buchi) system, again with cell recycle and withculture circulation, for a period of 24 hours. In this experiment the600 mL reactor was fed gas containing 15.8% H₂, 36.5% CO, 38.4% N₂ and9.3% CO₂ at a 1.4 minute gas retention time. The reactor pressure wasmaintained at 40 psig. Liquid medium containing excess salts, vitaminsand trace metals was fed at a liquid retention time of 4.8 hours, andthe cell retention time, obtained by passing effluent through a ceramichollow fiber filter, was 19.2 hours. The pH was 4.5, the agitation ratewas 1000 rpm and the liquid recirculation rate was 0.4 to 0.5 gpm. Underthese conditions, the CO conversion was 71.6% and the H₂ conversion was11.8%. The cell concentration was 7.1 g/L, ethanol was produced at 12.0g/L and acetate was produced at 2.7 g/L. The ethanol productivity was 60g/L·day.

In analyzing the parameters for ethanol production (Table 2), the ratioof pantothenate fed to the cell production was 294 μgpantothenate/g-cell produced. This level is far in excess of the minimumlevel required to cause ethanol production due to pantothenatelimitation. The rate of cobalt fed to the cell production was 735 μgcobalt/g cell produced, again a level that ensures the cobalt was fed inexcess. The ratio of H₂ fed to the sum of two times the CO converted andthree times the CO₂ converted was 0.3. The CO uptake rate was 0.67mmol/g cell·min., a level that again assures that excess CO is availableas the method of causing ethanol to be produced.

Example 7 Ethanol Production with Excess H₂ Present

A New Brunswick Scientific Bioflo® laboratory fermentation bioreactorwas operated as a straight through CSTR (without cell recycle) using C.ljungdahlii, strain C-01 ATCC 55988 for the production of ethanol fromCO, CO₂ and H₂ in the presence of excess H₂. The gas feed to the reactorcontained 77% H₂, 19% CO and 4% CH₄ (reference gas), fed at a gasretention time of 30 minutes. Liquid medium containing excess salts,vitamins and trace elements was fed to the reactor at a liquid retentiontime of 36 hours. The pH was 5.0 and the agitation rate was 1000 rpm.Under these operating conditions the CO conversion was 97-99% and the H₂conversion was 60-80%. The cell concentration was 0.8-1.0 g/L, theethanol concentration was 10 g/L and the acetate concentration was 3.3g/L. The ethanol productivity was 6.7 g/L·day.

In analyzing the parameters for ethanol production, the pantothenatefeed to cell production ratio was 900-1125 μg pantothenate/g cellproduced, thus assuring excess pantothenate was present. Similarly, thecobalt feed to cell production ratio was 991-1239 μg cobalt/g cellproduced, again assuring that excess cobalt was present. The specific COuptake rate was 0.28-0.35 mmol/g cell min, a level such that excess COwas not causing ethanol production. The ratio of the moles of H₂ fed tothe sum of 2 times the moles CO converted and three times the moles CO₂converted was 1.96, a ratio that is above 1.0, the level where excess H₂is present and thus could be controlling ethanol production. The H₂partial pressure in the exit gas was 0.70-0.87 atm, and the ratio of theH₂ partial pressure to CO₂ partial pressure in the exit gas was 65.Thus, the reactor was producing ethanol due to the presence of excessH₂.

In a second experiment, a high pressure AUTOKLAV™ reactor (Buchi) wasoperated as a CSTR with culture circulation and cell recycle using C.ljungdahlii, strain C-01 for the production of ethanol from CO, CO₂ andH₂ in the presence of excess H₂. The gas feed to the reactor contained81% H₂, 16% CO and 3% CH₄ (reference gas), fed at a gas retention timeof 2.21 minutes. Liquid medium containing excess salts, vitamins andtrace elements was fed to the reactor at a liquid retention time of 8.97hours. The cell retention time was 22.7 hours, the pH was 4.5 and theagitation rate was 800 rpm. Under these operating conditions the COconversion was 91.5% and the H₂ conversion was 43.4%. The cellconcentration was 5.5 g/L and the acetate concentration was 2.85 g/L.The ethanol productivity in the reactor was 215-240 g/L·day.

In analyzing the parameters for ethanol production, the pantothenatefeed to cell production rate was 46 μg pantothenate/g cell produced, alevel that may indicate pantothenate limitation. The cobalt feed to cellproduction ratio was 460 μg cobalt/g cell produced, a level whichassures that cobalt was not limiting. The specific CO uptake rate was1.68 mmol/g·cell·min, a level that could indicate that excess CO werepresent if it were not for the high H₂ uptake rate of 4.14mmol/g·cell·min, which indicates that substrate inhibition to the H₂conversion was not occurring. The ratio of the moles of H₂ fed to thesum of two times the moles CO converted and three times the moles CO₂converted was 5.67, a rate that is far above the required ratio of 1.0for excess H₂ to be present. The H₂ partial pressure in the exit gas2.61 atm, and the rate of H₂ partial pressure to CO₂ partial pressure inthe exit gas was 10.9. The reactor was thus producing ethanol as aresult of the presence of excess H₂.

A summary comparison of method parameters and results for Examples 3through 7 is shown in Table 2 below.

Example 8 Product Shift in C. ljungdahlii Strains ERI-2, C-01 and PETCUsing Medium Formulations

The methods of this invention can be applied to any of the C.ljungdahlii strains. Results from medium manipulation experimentsemploying strains ERI-2, C-01 and PETC are shown in Table 3 below. Thepurpose of these experiments was to demonstrate that each of the strainscan be shifted from acetic acid production to ethanol production merelyby manipulating the medium. Thus, a culture was fed excess nutrients(including pantothenate and cobalt) in order to produce acetic acid asthe dominant product, and then limited on pantothenate or cobalt toproduce ethanol as the dominant product. It should be emphasized thatthe only purpose of these experiments was to demonstrate that mediummanipulation can result in product shift for each of the strains. Thus,attaining high product concentrations and productivities was not a focusof these experiments.

The reactor was operated as a straight through CSTR (no cell recycle)for each of the culture experiments. The gas retention time wasnominally set at 50 minutes, the liquid retention time was nominally setat 40 hours and the agitation rate was nominally set at 1000 rpm. Theseconditions were chosen to allow comparisons of the strains, but not toachieve high productivities.

As noted in Table 3, strain ERI-2 was subjected to five changes inmedium which shifted the products back and forth from acetic acid as thedominant product to ethanol as the dominant product. Both pantothenatelimitation and cobalt limitation were demonstrated for ethanolproduction by this strain. Strain C-01 was shifted three times usingmedium manipulation, again with both pantothenate limitation and cobaltlimitation demonstrated as the mechanism for ethanol production. StrainPETC was shifted only once, with ethanol production due to cobaltlimitation. Each of the strains showed higher H₂ conversions whenproducing acetic acid, rather than ethanol, as the dominant product.This occurs because acetic acid is produced under mass transferlimitation (limiting the amount of gas to the culture), whereas ethanolis produced when limiting nutrients, and thus excess gas is suppliedwhich can negatively affect gas conversion. Small amounts of acetate arealways present in the product stream when the dominant product isethanol. However, when acetic acid is the dominant product, ethanol isusually not present in measurable concentrations. In shifting dominantproducts from ethanol to acetic acid by nutrient manipulation, it wasshown that it was very difficult to remove all traces of ethanol.Complete removal of ethanol occurred only after several weeks ofcontinued operation on acetic acid enhancing medium.

Example 9 Steady State Operation with and without Cell Recycle

The ultimate commercial goal of producing ethanol from CO, CO₂ and H₂ isto achieve high steady state concentrations of ethanol, while at thesame time, obtaining high ethanol to acetate product ratios and highproductivity. Steady state data for the production of ethanol fromCO-rich gas containing 20% H₂, 65% CO, 10% CO₂ and 5% CH₄ using C.ljungdahlii, strain C-01 in a straight through CSTR (no cell recycle)are shown in Table 4. In the table, GRT refers to the gas retention time(ratio of liquid volume to inlet gas flow rate), LRT refers to theliquid retention time (ratio of liquid volume to liquid flow rate), andXRT refers to the cell retention time (average amount of time cellsspend in the reactor). As is noted in the Table 4, ethanolconcentrations of 17.5 to 33 g/L were obtained, and the ethanolproductivity ranged from 14.4 to 21.1 g/L·day.

Similar results are shown for ethanol production from gas that is not asrich in CO. The gas used in the experiment using C. ljungdahlii C-01without recycle, for which results are reported in Table 5, contains 16%H_(z), 27% CO, 6% CO₂, and 51% N₂. Ethanol concentrations ranging from11 to 26 g/L were obtained with this gas, with 2.0 to 5.0 g/L acetatepresent as a secondary product. The ethanol productivity ranged from11.1-20.1 g/L·day. *The cell concentration is based upon dry cell weightin Table 5.

Finally, steady state data for the conversion of gas containing 50% H₂,45% CO and 5% CH₄ in a CSTR with cell recycle using C. ljungdahlii O-52(ATCC Accession No. 55989) are shown in Table 6 below. Ethanolconcentrations of 18 to 23.5 g/L and acetate concentrations of 3.0 to5.7 g/L were attained. The ethanol productivity ranged from 21.1 to 39.0g/L·day.

Example 10 High Ethanol Productivity in a CSTR with Cell Recycle andPressure

A high pressure AUTOKLAV™ reactor (Buchi) was operated as a CSTR withculture circulation and cell recycle using C. ljungdahlii, strain C-01for the production of ethanol from CO, CO₂ and H₂. The reactor wasoperated at 30 psig and fed gas containing 62% H₂, 31% CO and 5% C₂H₆.The gas retention time was 1.14 min (atmospheric pressure basis), withan actual gas retention time of 3.5 min. Liquid medium containing excesssalts, vitamins and trace metals was fed to the 600 mL reactor at aliquid retention time of 3.6 hours. The pH was 4.5 and the agitationrate was 825 rpm. Under these conditions, the cell concentration was 8g/L, the CO conversion was 90% and the H₂ conversion was 40%. Theproduct stream contained 20 g/L ethanol and 2.75 g/L acetate. Theethanol productivity was 150 g/L·day.

In another high pressure AUTOKLAV™ reactor (Buchi) operated as a CSTRwith culture circulation and cell recycle using C. ljungdahlii, strainC-01, the reactor was operated at 6 atm (75 psig) and fed syngascontaining 55% H₂, 30% CO, 5% CH₄ and 10% CO₂. The gas retention timewas 1 min (atmospheric pressure basis), with an actual gas retentiontime of 6.0 min. Liquid medium containing excess salts, vitamins andtrace metals was fed to the reactor at a liquid retention time of 1.62hr. The cell retention time was 24 hr, the pH was 4.5 and the agitationrate was 800 rpm. Under these conditions, the cell concentration was 2.0g/L, the CO conversion was 95% and the H₂ conversion was 60%. Theproduct stream contained 25 g/L ethanol and 3 g/L acetate. The ethanolproductivity was 369 g/L·d.

Example 11 Start-Up from Stock Culture with Excess H₂ Present

Start-up using a batch inoculum from stock culture ensures a healthyinoculum free from contaminants, but is not always successful as aninoculation procedure because of the rather low cell density employed,especially if the method parameters such as gas rate and agitation rateare pushed upward too rapidly just after inoculation.

Start-up using batch inoculum from stock culture is discussed in thisexample. To prepare the stock cultures for inoculation of the reactor,cultures of C. ljungdahlii, strain C-01 (ATCC Accession No. 55988) weregrown up in 150 mL serum bottles on CO, CO₂ and H₂ in a rich mediumcontaining 1 g/L yeast extract and 1 g/L trypticase, in salts andvitamins. The vitamin concentration employed was 0.4 mL/L medium of anaqueous solution containing 50.5 mg/L calcium pantothenate, 20.6 mg/Ld-biotin and 50.6 mg/L thiamine HCl. Bottles were incubated at 37° C. ina shaker incubator. The cultures were grown to the exponential growthphase, as determined by visual inspection. With each inoculation,approximately 90 mL of stock culture were transferred from serum bottlesto 1 liter of medium, representing 9% by volume inoculation. Asuccessful inoculation is described below. The outlined procedure can berepeated several times to obtain a successful inoculation.

In obtaining a successful inoculation, 90 mL/L of inoculum were added toa 1 liter batch of basal medium (shown in Table 1) containing 0.4 mL/Lvitamins and salts (t=0). The agitation rate was 240 rpm, the pH was5.3, the temperature was 38.5° C. and the gas retention time (continuousgas flow) was 110 minutes. The gas feed contained 62% H₂, 31% CO and 7%C₂H₆. After 13 hr (t=13 hr) some CO conversion was noted, and at t=23 hrthe agitation rate was increased from 240 rpm to 300 rpm. The gasretention time was decreased to 100 minutes at t=27 hr, and a furtherdecrease in gas retention time was made at t=46 hr. The agitation ratewas also increased in 100 rpm increments at t=28 hr, 59 hr, 72 hr and 85hr.

By t=110 hr, the system was operating with a gas retention time of 80minutes and an agitation rate of 600 rpm. The cell concentration was 0.5g/L and the CO conversion was 35%. There was still no H₂ conversion, butsmall amounts of ethanol and acetate (˜1 g/L each) had accumulated inthe batch culture broth. The efforts up until this time emphasized cellgrowth in the reactor.

Medium flow using the same concentrations as in basal medium was startedat a rate of 0.4 mL/min at t=120 hr. A program of nominal increases ingas rate, agitation rate and medium rate was then initiated whilecarefully maintaining the system under excess H₂. By t=210 hr, theethanol concentration was 17 g/L, the acetate concentration was 1 g/L,the cell concentration was 1.6 g/L, the CO conversion was nearly 100%and the H₂ conversion was 90%. The ethanol productivity reached 11.4g/L·day.

A program of gradual gas rate increases was again started. Concurrentvitamin (see Table 1) increases were made to bring the vitamin additionrate to 0.7 mL/L medium. By t=610 hr, the reactor was producing 20 g/Lethanol and about 2 g/L acetate. The CO conversion was nearly 100% andthe H₂ conversion was 85%. The ethanol productivity reached 14 g/L·day.

Example 12 Start-Up Using Inoculum from Existing CSTR

The start-up of a CSTR using continuous inoculum from an existing CSTRis much faster and is more dependable than a start-up from batch bottlesof stock culture. A CSTR containing Isolate C. ljungdahlii, strain C-01(ATCC Accession No. 55988), that had nearly ceased ethanol productionand was producing 2-3 g/L ethanol, 7-8 g/L acetate and about 0.3 g/Lbutanol as the liquid phase products, was restarted using a continuousinoculum from an existing CSTR.

The CSTR from which the inoculum was taken was producing about 17 g/Lethanol and 1-2 g/L acetate, while operating at a gas retention time of25 minutes, a liquid retention time of 32 hours, an agitation rate of650 rpm, a temperature of 38.5° C. and pH 4.66. The cell concentrationwas 1.7 g/L, the CO conversion was essentially 100% and the H₂conversion was 85%.

Continuous inoculum addition was started (t=0), and at this time, theagitation rate was reduced to 500 rpm and the gas retention time was setat 38 minutes. Effluent from the productive reactor (0.5 mL/min) servedas the continuous inoculum for the CSTR being inoculated, withcontinuous inoculation occurring over a period of several hours. By t=5hr (5 hr after the onset of continuous inoculation), gas conversion wasnoted, and the agitation rate was increased to 700 rpm. The continuousinoculum was turned off at t=28 hr. The gas conversions improvedsteadily, allowing steady increases in gas rate (lowered gas retentiontimes) and an agitation rate increase to 750 rpm. By t=30 hr, the COconversion was 95% and the H₂ conversion was 80%. The ethanolconcentration was 13 g/L and acetate concentration was 1.5 g/L, and itsteadied at 1.4 g/L for well over 100 hours. During this time period,the ethanol productivity was 10 to 15 g/L·day.

Example 13 Recovery from Severe Method Upset

A CSTR with cell recycle containing C. ljungdahlii, strain C-01 beingcontinuously fed gas and liquid nutrients and producing 15-35 g/Lethanol and 0-5 g/L acetate at a steady state (e.g., Example 1) is upsetdue to unforeseen changes in method conditions, e.g., mechanicalproblems in the reactor. Upset to the reactor system can either beminor, such as a brief increase in the gas rate which causes short-termsubstrate inhibition, or major, such as a longer term increase in thegas rate which eventually leads to increased acetic acid production andmore severe molecular acetic acid product inhibition.

Short-term upsets are easily corrected by merely readjusting the upsetparameter (for example, lowering the gas rate to its original level) andmonitoring the progress of the reactor to assure that the upset has notled to a longer-term problem.

However, acetic acid product inhibition is a more severe problem. Ifexcess molecular acetic acid is produced by the culture as a result oflong term substrate inhibition, excess nutrient addition, CO₂accumulation or mechanical problems of many types, the problem that ledto the excess acetic acid must first be corrected. The excess aceticacid, which quickly leads to product inhibition, is then cleared fromthe system by increasing the liquid rate to wash the acetic acid (andunfortunately ethanol) from the system. Once the acetate level is below3-5 g/L, the liquid rate is reset and the reactor is placed back undereither excess H₂ feed, or vitamin or cobalt limitation (with or withoutcell recycle). Bringing the reactor back involves reducing the gas rateto avoid substrate inhibition and/or agitation rate before cell washoutand lysis takes place. The agitation rate or gas rate is then increased,as described in Example 1.

In one specific example, a CSTR with cell recycle containing C.ljungdahlii, strain C-01 that was producing ethanol and acetic acid fromCO, CO₂ and H₂ began producing acetic acid in response to a mechanicalproblem. The 2100 mL reactor was fed gas containing 62% H₂, 31% CO and7% C₂H₆ at a gas retention time of 15 minutes, and was operating with anagitation rate of 600 rpm and a pH of 4.86. The liquid retention timewas 23 hours and the cell retention time was 68 hours. B-vitaminsolution (an aqueous mixture of 50.5 mg/l calcium pantothenate, 20.6mg/L d-biotin and 50.6 mg/L thiamine HCl) was present in the liquidnutrient medium containing salts and vitamins at a concentration of 0.4mL vitamin solution per liter of medium (see Table 2). The ethanolconcentration fell to 7 g/L, while the acetate concentration rose to 7g/L, conditions that are neither stable for operating the reactor noreconomical for ethanol production. The cell concentration was 2.4 g/L,the CO conversion was 85% and the H₂ conversion was 25%.

The strategy used in recovering the reactor consisted of firstdramatically reducing the gas feed rate to the reactor, followed bygradual recovery of the reactor in the presence of excess H₂. The liquidrate to the reactor was not reduced to clear product inhibition in thisexample because the acetate concentration was not exceedingly high.Instead, the acetate concentration was allowed to more gradually drop tonon-inhibiting levels with the reduction in gas flow rate and subsequentoperation in the presence of excess H₂. The specific procedure inrecovering the reactor is discussed below.

Cell recycle was turned off and the gas rate was dramatically reduced by70% to a gas retention time of 62 minutes, while only slightly adjustingthe liquid retention time from 23 to 30 hours (t=0). The vitaminconcentration in the medium was not changed. With this change in gasrate the CO conversion increased to 98% and the H₂ conversion increasedto 80%. More importantly the system had excess H₂ present, as evidencedby the decrease in CO₂ in the outlet gas from 19 to 5%. With the onsetof excess H₂, the acetate concentration fell while the ethanolconcentration increased. At t=66 hr (66 hr after turning off cellrecycle), for example, the acetate concentration had fallen to 4 g/L andthe ethanol concentration had risen slightly to 7.5 g/L.

The presence of excess H₂ (and the lowered acetate concentration)permitted subsequent increases in gas rate, first slowly and then at afaster rate. By t=215 hr the gas retention was 29 minutes, the ethanolconcentration was 12 g/L and the acetate concentration was 3 g/L. Theethanol productivity was 8 g/L·day. CO₂ was present in the outlet gas at6%, the CO conversion was 98% and the H₂ conversion was 80%. By t=315hr, the ethanol concentration was 16 g/L and the acetate concentrationwas 4 g/L, again with good gas conversions, and a gas retention time of20 minutes. The ethanol productivity was 11 g/L·day. By t=465 hr, theethanol concentration had reached 20 g/L, with 3.5 B 4 g/L acetate alsopresent. The ethanol productivity was 16 g/L·day. The gas retention timehad been dropped to 16 minutes, with CO and H₂ conversions of 95 and73%, respectively. These conditions were maintained for nearly 200 hoursof continuous operation, demonstrating that the reactor system hadrecovered its ability to produce ethanol and had essentially retainedthe previous operating conditions.

Example 14 Ethanol Production Method with Oversupply of CO

A simple experiment was performed in a continuous high pressure stirredtank reactor with cell recycle to demonstrate the shift from acetic acidproduction to ethanol production due to the presence of high COconcentrations. Prior to this experiment the reactor containing C.ljungdahlii, strain C-01 was operated at a pressure of 20-25 psig andfed gas containing 57% H₂, 36% CO and 7% C₂H₆. The gas retention timewas less than 2 minutes, the liquid retention time was 38 hours, thecell retention time was 28 hours, the agitation rate was 600 rpm and thetemperature was 38° C. Under these conditions the CO conversion wasvariable and averaged 85%, and the H₂ conversion was variable andaveraged 20%. The cell concentration was about 2.5 g/L, and the productstream contained 9 g/L ethanol and 3 g/L acetate.

As a first step in preparing for the test, the gas retention time wasincreased in order to ensure that excess CO was not present. Thepressure was maintained at 23-24 psig. The pH was followed long enoughto ensure that it was stable in the normal operating range of 4.5-4.6.Pure CO was then blended with the regular feed gas to yield a gas feedof 47% H₂, 47% CO and 6% C₂H₆ at a gas retention time of 2.3 minutes.The reactor pH, exit gas composition, and product stream were thenmonitored with time.

Table 7 shows the pH changes and product compositions with time afterthe addition of extra CO to the system. Thirty minutes after the COaddition, the reactor pH had increased to 5.25 and the culture hadshifted 1.54 g/L (0.0257 mole/L) acetate to 1.12 g/L (0.0243 mole/L)ethanol. The pH increase occurred as a result of the free acetic acidbeing converted to ethanol. Accompanying this change was a decrease inCO conversion from 91% to 71%. In decreasing the culture circulationrate from 0.4 gpm to 0.15 gpm, the reactor pH fell, but the ethanol andacetate concentrations held.

Fifty minutes after CO introduction the ethanol concentration was 11.29g/L and the acetate concentration was 1.75 g/L. At this time, the excessCO was turned off and the ethanol concentration and pH began to fall,and the acetate concentration began to rise. The decrease in pH was dueto the conversion of ethanol to molecular acetic acid. Theethanol-acetic acid shift through oversupply of CO is thus reversible.

Example 15 Water Recycle to Minimize Acetate Production

The recycle of method water back to the fermentation bioreactor afterdistillation to recover ethanol is essential to minimize effluentproduction, and to maximize the yield of ethanol from the reactor, andto limit the acetic acid production. Distillation has been found to bethe most economical method for concentrating 15-35 g/L ethanol obtainedfrom the reactor to 95% ethanol. Adsorption with molecular sieves isthen used to further concentrate the ethanol to the desiredconcentration. In performing the distillation, 95% ethanol in water isproduced as the overhead product. Water is generated as the bottomsproduct during distillation. The bottoms product contains acetic acidfrom the reactor produced during fermentation (3-5 g/L acetate) and anynutrients not used up during fermentation or destroyed by the heat ofdistillation, such as trace metals and other minerals. The recycle ofnutrients minimizes the quantity of effluent that must be treated aswell as the quantity of nutrients that must be subsequently added to thefermentation bioreactor. The recycle of acetate prevents the formationof further acetic acid by establishing equilibrium between the ethanoland acetic acid. Thus, no net acetic acid is produced with waterrecycle. Recycle of more than 3-5 g/L acetate can result in acetic acidinhibition in the reactor. Thus, as a result of water containing acetaterecycle, the substrate CO, CO₂ and H₂ can be converted to ethanol as theonly product.

Table 8 shows results for the fermentation of gas containing 50% CO, 45%H₂ and 5% CH₄; using C. ljungdahlii, strain O-52 with water recycle. Inthese experiments, the permeate from hollow fiber filtration used forcell recycle was sent to distillation. After removing ethanol, the waterwas filtered with a 0.2 micron filter to remove any precipitatedby-products. The fraction of water recycled compared to the total water(as medium) fed to the reactor in these experiments ranged from 25-100%.The experiment with 100% water recycle lasted for nearly 500 hours orabout 20 liquid retention times. As is noted in the results with 100%water recycle, no net acetic acid was produced. In fact, a small amountof acetic acid was eventually consumed. The ethanol productivity rangedfrom 12 to 27 g/L·day.

Example 16 Two-Stage CSTR System with Pantothenate Feed to the GrowthStage

The proper pantothenate feed to the growth stage is a variable that mustbe optimized. Typical results from a Growth Stage Reactor using C.ljungdahlii C-01 were described in Examples 11 and 12, with theexception that a bit more acetic acid would be produced in this reactorsince additional pantothenate or cobalt is fed to the Growth Stage toensure a healthy and stable culture. The vitamin concentration employedwas 0.7-0.8 mL/L medium of an aqueous solution containing 50.5 mg/Lcalcium pantothenate, 20.6 mg/L d-biotin and 50.6 mg/L thiamine HCl. TheProduction Stage CSTR with cell recycle is fed effluent from the growthstage reactor and produces ethanol as the predominant product. Thepantothenate concentration fed to this reactor is much lower than in theGrowth Stage, only 0.1-0.2 mL total vitamins/L medium of the aqueoussolution containing 50.5 mg/L calcium pantothenate, 20.6 mg/L d-biotinand 50.6 mg/L thiamine HCl. The gas retention time in this ProductionStage was 11-30 minutes, the liquid retention time was about 20 hours,the cell retention time was 30-50 hours, and the agitation rate was800-900 rpm. The pH was 5.0 and the temperature was 38° C. Once thereactor reached steady state, the gas retention time was held constantat 11 minutes, the liquid retention time was set at 19 hours, the cellretention time was constant at 37 hours and the agitation rate was 900rpm. The CO conversion averaged 96% and the H₂ conversion averaged 60%.The ethanol concentration steadied at 25-30 g/L, with about 3 g/Lacetate also present. The ethanol productivity was 31.6-37.9 g/L·day.

Example 17 Regulating the Fermentation Parameters to Avoid Acclimationto Low Limiting Calcium Pantothenate

The acclimation of the culture in the fermentation bioreactor to lowlimiting calcium pantothenate concentration is avoided by regulating thefermentation parameters (gas rate, liquid rate, agitation rate, H₂partial pressure) while avoiding major changes in nutrients, but insteadmaintaining a relatively constant nutrient feed concentration, asfollows.

During start-up of a laboratory New Brunswick Scientific Bioflo® CSTR,C. ljungdahlii, strain C-01 was fed a liquid nutrient stream containingvitamins, trace minerals and salts necessary to provide nutrition to theculture. The pantothenate concentration in the nutrient medium was 20μg/L, a concentration that when coupled with the slow rate of mediumfeed ensures that there is more than 100 μg calcium pantothenate fed pergram of cells produced (excess pantothenate) because of low cellproduction in the bioreactor. Similarly the cobalt concentration in themedium was 1 ppm, a concentration that ensures cobalt is also present inexcess. Instead, the H₂ partial pressure in the exit gas was kept inexcess of than 0.55 atmospheres by feeding a gas containing no CO₂,63.3% H_(z), 31.4% CO and 5.3% C₂H₆, thus yielding a ratio of H_(2 fed)(2 CO_(converted) and 3CO_(2 converted)) of more than 1 and by carefullyregulating the gas feed rate and agitation rates to achieve greater than95% CO conversion and greater than 80% H₂ conversion. As these highconversions are attained with time, the cell concentration builds froman initial level of near 0 g/L to about 1.5 g/L.

Since the pantothenate concentration is held constant during thisstart-up, the μg pantothenate per gram of cells produced graduallydecreases until it is less than 15 μg pantothenate/g cell produced, acondition which is then pantothenate limited. The system thus grows intopantothenate limitation. High ethanol:acetate product ratios areattained throughout the start-up by excess H₂. Alternatively the reactoris allowed to produce acetic acid during the early stages of start-up,with the product ratio later brought under control through pantothenatelimitation.

Example 18 Limiting Cobalt to the Reactor

C. ljungdahlii, strain ERI-2 was fed 62 to 3500 μg cobalt/g cellproduced during acetic acid production from CO, CO₂ and H₂, a conditionwhere the reactor was not limited on cobalt (or any other limitationexcept for the ability to transfer gas to the culture), and no ethanolwas found in the product stream. During limitation on cobalt for ethanolproduction from CO, CO₂ and H₂ , C. ljungdahlii strain C-01 was fed 33to 48 μg cobalt/g cells produced, while maintaining all other nutrientsin excess. Under these conditions, strain C-01 produced 18 to 26 g/Lethanol and about 4 g/L acetate.

Example 19 Avoiding Acclimation to Low Limiting Cobalt Concentration

Acclimation to low limiting cobalt concentration is avoided byregulating the fermentation parameters (gas rate, liquid rate, agitationrate, CO₂ content) while avoiding major changes in nutrients, butinstead maintaining a relatively constant nutrient feed concentration,as follows.

During start-up of a laboratory New Brunswick Scientific Bioflo® CSTR,C. ljungdahlii, strain C-01 was fed a liquid nutrient stream containingvitamins, trace minerals and salts necessary to provide nutrition to theculture. The cobalt concentration in the nutrient medium was 75 ppb, aconcentration that when coupled with the slow rate of medium feedensures that there is more than 50 μg cobalt fed per g of cells produced(excess cobalt) because of low cell production in the bioreactor.Similarly the pantothenate concentration in the medium was 20 μg/L, aconcentration that ensures pantothenate is also present in excess.Instead, the H₂ partial pressure in the exit gas was kept in excess of0.55 atmospheres by feeding a gas containing large quantities of H₂ andno CO₂, and by carefully regulating the gas feed rate and agitationrates to achieve greater than 95% CO conversion and greater than 80% H₂conversion. As these high conversions are attained with time, the cellconcentration builds from an initial level of near 0 g/L to about 1.5g/L. Since the cobalt concentration is held constant during thisstart-up, the μg cobalt per g cells produced gradually decreases untilit is less than 50 μg cobalt/g cell produced, a condition which is thencobalt limited. The system thus grows into cobalt limitation. Highethanol yields are attained throughout the start-up by employing excessH₂ in the feed. Alternatively the reactor is allowed to produce aceticacid during the early stages of start-up, with the product ratio laterbrought under control through cobalt limitation.

Example 20 Oversupplying Hydrogen

During operation of a laboratory AUTOKLAV™ reactor (Buchi) operated as aCSTR with liquid recirculation and cell recycle, C. ljungdahlii wasoperated with excess vitamins, trace minerals and salts necessary toprovide nutrition to the culture. The reactor was operated with excessH₂ present in the feed gas such that the ratio of the moles of H₂ fed tothe sum of two times the moles of CO converted and three times the molesof CO₂ converted was 5.67. If this ratio were not greater than 1.0,excess H₂ cannot be present in the reactor and ethanol production due tothe presence of excess H₂ cannot occur. Furthermore, the H₂ partialpressure in the exit gas was 2.61 atm, a level that exceeds therequirement of 0.4 atm for ethanol production due to excess H₂. Finally,the ratio of H₂ partial pressure to CO₂ partial pressure in the exit gaswas 10.88, a level which is greater than 3.0 and assures that enough H₂is present to utilize all of the available carbon. Under theseconditions the reactor produced nearly 26 g/L ethanol and less than 3g/L acetate. The ethanol productivity was more than 200 g/L·day. If anyof these above criteria are not met, the reactor cannot produce ethanoldue to excess H₂ being present. Another aspect of H₂ abundance is thatit results in additional reduced ferredoxin by oxidation throughhydrogenase.

Example 21 Alleviating CO Substrate Inhibition

A laboratory New Brunswick Scientific Bioflo® CSTR operating at anagitation rate of 800 rpm shows an outlet CO concentration of 10% whenit had been previously operating with only 5% CO in the gas outlet. Bydecreasing the agitation rate to 600 rpm, CO inhibition was removed andthe outlet CO concentration returned to 5%. This results in increased H₂uptake, a necessary condition to efficiently utilize all of the gas fedto the reactor.

Example 22 Mass Transfer

As an example of excess mass transfer leading to ethanol production,consider a laboratory CSTR with cell recycle containing C. ljungdahlii,strain ERI-2 operating without nutrient limitation or excess H₂ or CO inthe feed gas. That is, pantothenate is fed at a rate of more than 100 μgcalcium pantothenate per gram of cells produced and cobalt is fed at arate of more than 100 μg per gram of cells produced. H₂ is present inthe exit gas at about 0.2 atm and the specific CO uptake rate is lessthan 0.3 mmol CO/g cells·min. The agitation rate is 800 rpm. Under theseconditions the culture produces only acetic acid (no ethanol present inthe product stream). If the agitation rate is increased quickly to 900rpm or the gas rate is increased by about 10%, ethanol is observed inthe product stream, until the cell concentration increases in order touptake the gas or until the culture dies due to substrate inhibition.

Example 23 Controlling Acetic Acid Product Inhibition

In a laboratory CSTR which is producing 8 g/L acetic acid and 10 g/Lethanol, the liquid retention time is reduced from 24 hours to 12 hoursfor a period of 36 hours in an attempt to wash out the excess aceticacid from the reactor which is limiting the ability of the culture toproduce more ethanol. All other reactor operating and nutrientconditions are held constant. After this period of time, the liquidretention time is returned to 24 hours and a product stream containing 3g/L acetate and 15 to 25 g/L ethanol results. Several attempts inreducing the liquid retention time are required to clear the productinhibition. Alternatively, H₂ is added to the gas feed to allow excessH₂ control, since excess CO₂ can also lead to acetic acid production infavor of ethanol. These modifications prevent excess acetic acidproduction, and thus prevent a poor product ratio, and a low ethanolproductivity. Thereafter, the use of excess H₂ in the feed gas orlimiting liquid phase nutrient concentration is resumed.

Example 24 Oversupplying Carbon Monoxide

C. ljungdahlii, strain ERI-2 when fed excess nutrients (pantothenate andcobalt in excess) and without an abundance of H₂ in the feed gas had aspecific CO uptake rate of 0.23 to 0.48 mmol/g·min., and no ethanol wasfound in the product stream. However, when C. ljungdahlii, strain C-01was similarly fed excess nutrients without an abundance of H₂, in thefeed gas, but was under a condition where an oversupply of CO wascausing ethanol production, the specific CO uptake rate was 0.67 to 1.34mmol/g·min. Under these conditions the culture produced 9.9 to 12.0 g/Lethanol and 2.6 to 2.7 g/L acetate.

Example 25 Controlling Product Ratios with Cell Purge

A gaseous substrate (30% CO, 15% H₂, 10% CO₂, 45% N₂) fermentation takesplace in a CSTR (pH=5.0, Temperature=38° C., Pressure=20 psig) utilizingC. ljungdahlii, strain C-01, with cell recycle (cell retention time=40hours and the liquid retention time=6 hours) and the culture is notlimited in growth by cobalt, calcium pantothenate, or any othernutrient. As the culture grows, a cell density is attained such that thespecific uptake (mmol CO per gram of dry cells per minute) is below 0.5and acetic acid is produced preferentially to ethanol. To prevent thisoccurrence, the cell purge rate is increased to prevent an increase incell density, such that the steady concentration of cells is kept lowenough to maintain a specific uptake higher than 0.5 mmol CO per gramdry cells per minute. In doing so, the cell retention time is reduced tobetween 6 and 25 hours.

TABLE 1 Ethanol Production Medium Component Quantity Per Liter 2 g/lFeCl₂•4H₂O 10 mL 85% H₃PO₄ 0.05 mL MPFN Trace Metals^(a) 20 mL(NH₄)₂HPO₄ 0.60 g NH₄Cl 2.00 g NaCl 0.20 g KCl 0.15 g MgCl₂•6H₂O 0.50 gCaCl₂•2H₂O 0.20 g Cysteine HCl•H₂O 0.25 g Vitamins solution^(b)variable^(c) ^(a)MPFN Trace Metals contains (per liter of solution): 10mL of 85% H₃PO₄, 0.10 g of ZnSO₄•7H₂0, 0.03 g of MnCl₂•4H₂O, 0.3 g ofH₃BO₃, 0.20 g of CoCl₂•6H₂O, 0.02 g of CuCl₂•H₂O, 0.04 g of NiCl₂•6H₂O,0.03 g of NaMoO₄•2H₂O, 2.00 g of FeCl₂•4H₂O, 0.01 g of Na₂SeO₃, and 0.10g of Na₂WO₄•2H₂O ^(b)Vitamins solution contains 20.6 mg/L d-biotin, 50.6mg/L thiamine HCl and 50.5 mg/L d-pantothenic acid, calcium salt^(c)Varies considerably from 0.3-0.5 mL at inoculation to as much as0.7-0.8 mL at high gas rates

TABLE 2 Summary Comparison of Method Parameters and Results for Examplesof Control Methods Pantothenate Cobalt Product Supplied Supplied H₂Specific Concentrations Ethanol (μg (μg Cobalt/ Partial CO UptakeExample Controlling Ethanol Acetate Productivity pantothenate/ g cellH_(2 Fed) Pressure in (mmol/g · No. Mechanism (g/L) (g/L) (g/L · day) gcell produced) produced) (2CO_(conv) + 3CO_(2 conv)) Exit Gas (atm) cell· min) 3 Mass Transfer 0 10-13 0 1575-3150 1734-3468 0.46 0.06-0.070.275-0.48  3 Mass Transfer 0 10-14 0 2250-3600 62-99 0.875 0.11-0.200.33-0.40 4 Pantothenate 15-19 1.5 11.5-14.5 18-24 5000-6660 1.030.55-0.64 0.23-0.30 4 Pantothenate 18 3 17.4 8.1 3960 1.14 0.60-0.650.33 5 Cobalt 26 4 15.6 15.2 33 0.94 0.63 0.37 5 Cobalt 18 4 27.0 85.747.6 1.03 0.60 0.50 6 Excess CO 9.9 2.6 29.0 97 83.6 1.09 1.6 1.34 6Excess CO 12.0 2.7 60.0 294 735 0.30 0.6 0.67 7 Excess H₂ 10.0 3.3 6.7 900-1125  991-1239 1.96  0.7-0.87 0.28-0.35 7 Excess H₂ 25.96 2.85215-240 46 460 5.67 2.61 1.68

TABLE 3 Summary of Product Shift with Clostridium ljungdahlii StrainsGas Product Cell Con- Concentration C. ljungdahlii Medium Conc.* versionAce- Strain Limitation (g/L) CO H₂ Ethanol tate ERI-2 Acetic Acid 1.1 9080 0 10 Enhancing ERI-2 Pantothenate 0.3 88 20 2.5 0 Limitation ERI-2Acetic Acid 0.55 90 85 1.0 5.5 Enhancing ERI-2 Pantothenate 0.5 90 20 101.0 Limitation ERI-2 Acetic Acid 0.8 100 93 1 7 Enhancing ERI-2 Cobalt1.3 80 20 9 3 Limitation C-01 Acetic Acid 1.2 96 90 1 8 Enhancing C-01Pantothenate 0.8 60 30 4 0 Limitation C-01 Acetic Acid 1.2 96 90 <1 9Enhancing C-01 Cobalt 2.5 80 20 17 2 Limitation PETC Acetic Acid 0.8 6555 2 10 Enhancing PETC Cobalt 1.0 95 55 8 1 Limitation *Dry cell weightbasis

TABLE 4 Steady State Data for the Conversion of CO-Rich Gas to EthanolUsing C. ljungdahlii, Strain C-01 Gas Ethanol Con- Products Produc-Agitation Cell version (g/L) tivity GRT LRT Rate Conc* (%) Ace- (g/L ·(min) (hr) (rpm) (g/L) CO H₂ Ethanol tate day) 13 32.4 700 2.44 91 5721.6 3.9 16.0 11.93 25.7 750 2.51 92 54 20.6 3.6 19.2 12.67 25.6 7502.60 93 61 18.7 4.7 17.6 10 24.5 750 2.75 92 43 20.4 6.1 20.0 11.54 23.8750 2.65 92 40 20.4 5.3 20.6 12.10 23.8 750 2.77 88 18 21 3.1 21.1 13.823.8 750 2.70 90 25 18 2.5 18.2 12.7 23.8 750 2.70 92 35 20 3.8 20.213.3 24.0 800 2.70 85 10 17.5 5.0 17.5 14.81 31 750 2.50 92 30 25 2.519.4 16.9 31 750 3.60 90 18 23 3.0 17.8 18.5 33 750 2.60 94 50 24 3.517.5 17.2 34 750 2.50 91 40 24 3.5 16.9 18.5 34 750 2.30 95 63 23 4.016.2 19.2 40.6 750 2.70 94 50 28.5 4.0 17.4 19.0 55 750 2.70 94 20 334.0 14.4 *Dry cell weight basis

TABLE 5 Steady State Data for the Conversion of Gas Containing 27% CO,16% H₂, 51% N₂ to Ethanol Using C. ljungdahlii, Strain C-01. No CellRecycle Gas Ethanol Con- Products Produc- Agitation Cell version (g/L)tivity GRT LRT Rate Conc* (%) Ace- (g/L · (min) (hr) (rpm) (g/L) CO H₂Ethanol tate day) 8.89 23.8 750 2.3 84 57 11 2.5 11.1 8.3 23.8 900 2.689 55 12 2.0 12.1 8.3 27.7 900 2.7 89 47 15 3.0 13.0 7.1 33.3 900 3.0 8637 19 3.0 13.7 7.4 33.3 900 3.0 87 40 19.5 3.0 14.1 6.34 33.3 900 3.0 8637 21 3.5 15.1 6.18 33.3 900 3.0 86 41 20.9 3.1 15.1 5.72 34.3 900 3.085 40 22.1 3.8 15.5 5.12 33 900 3.7 85 40 25.0 4.0 18.2 4.59 33 900 4.183 33 25 3.5 18.2 4.59 29 900 4.0 80 35 23 4.0 19.0 4.76 29 900 3.9 9035 19 5.0 15.7 4.25 28 900 4.2 80 30 23 3.0 19.7 5.5 37 900 3.4 84 40 233.0 14.9 5.26 31 900 3.8 84 50 23 3.0 17.8 5.71 31 900 3.7 80 28 26 3.520.1 6.25 31 900 3.75 82 30 25.5 3.0 19.7 6.66 31 900 3.6 86 64 22 4.017.0

TABLE 6 Steady State Data for the Conversion of Gas Containing 50% H₂,45% CO and 5% CH₄ to Ethanol Using Isolate O-52 in a CSTR with CellRecycle Gas Ethanol Con- Produc- Cell version Products tivity GRT XRTLRT Conc* (%) (g/L) (g/L · (min) (hr) (hr) (g/L) CO H₂ Ethanol Acetateday) 12.5 46.4 23.2 3.8 96.3 81.2 20.4 4.4 21.1 9.7 43.2 17.3 4.9 86.749.9 21.1 3.5 29.3 9.2 43.2 17.3 4.6 89.4 64.5 20.5 5.1 28.4 7.5 43.217.3 5.0 81.8 42.1 22.2 3.7 30.8 9.2 49.4 17.3 4.6 85.3 52.1 21.1 4.429.3 8.4 46.0 16.1 4.5 85.2 61.4 20.8 5.1 31.0 6.8 54.3 16.3 4.7 84.757.7 23.4 5.7 34.5 7.2 54.3 16.3 4.0 83.1 55.2 19.0 4.4 28.0 7.4 54.316.3 5.0 86.6 66.7 21.9 5.5 32.2 6.4 55.6 16.7 5.6 83.3 53.1 23.5 4.933.8 6.2 41.6 14.5 5.7 82.5 55.0 20.1 5.0 33.3 6.0 41.6 14.5 6.0 82.550.0 21.5 3.0 35.6 6.0 34.2 12.0 5.7 84.0 56.0 19.5 4.5 39.0 5.7 34.212.0 5.7 81.0 45.0 18.0 4.5 36.0 *Dry cell weight basis

TABLE 7 pH and Liquid Sample Analyses in Shifting Acetate to Ethanol inthe Presence of Excess CO Cell Conc.* Ethanol Acetate Butanol Time pH(g/L) (g/L) (g/L) (g/L) 0 4.69 2.4 10.3 3.1 0.3 30 5.28 11.4 1.5 0.3 355.28 2.4 11.6 1.6 0.3 50 4.98 11.3 1.8 0.3 80 4.73 2.4 10.9 2.9 0.3*Based on dry cell weight

TABLE 8 Data for Gas Fermentation by Isolate O-52 with Cell and WaterRecycle Eth- anol % Gas Products Pro- Wa- Con- Net duc- ter Cell versionEth- Ace- Ace- tivity Time Re- Conc.* CO H₂ anol tate tate (g/L · (hr)cycle (g/L) (%) (%) (g/L) (g/L) (g/L) day)  0-75 25 2.1 95 68 12 4 4 12 75-193 50 2.1 95 75 15 6 5 15 193-462 75 2.1 92 60 17 5 4 17 462-554 501.6 85 30 17→13 5 3 12-16 554-669 75 2.6 92 75 13→19 5 3 12-18 669-943100 3.0 92 70 23 6 3 23  943-1087 100 3.0 92 60 23 6 0 23 1087-1232 1002.7 92 60 23 6 −0 23 1232-1375 100 3.0 91 60 27 6 −1 27 1375-1534 1003.5 88 35 23 5 0 23 *Dry cell weight basis

All published documents are incorporated by reference herein. Numerousmodifications and variations of the present invention are included inthe above-identified specification and are expected to be obvious to oneof skill in the art. Such modifications and alterations to thecompositions and methods of the present invention are believed to beencompassed in the scope of the claims appended hereto.

We claim:
 1. A continuous method for producing ethanol comprising:culturing in a fermentation bioreactor a strain of Clostridiumljungdahlii bacteria in a continuously fed liquid nutrient medium toprovide a fermentation broth; supplying vitamin solution to fermentationbroth, supplying to said fermentation bioreactor a gaseous substratecomprising carbon monoxide; maintaining a specific rate of CO uptake insaid fermentation bioreactor at an amount of 0.3 to 2 mmol CO/gram drycells weight of bacteria per minute in the fermentation broth after thebacteria achieve a stable cell concentration in the fermentationbioreactor; and removing the fermentation broth from the fermentationbioreactor, distilling ethanol from the broth, and recovering theethanol; wherein the bacteria produce both ethanol and acetate in aratio of ethanol to acetate ranging from 1:1 to 20:1; and wherein theratio of acetic acid to ethanol is less than 0.8:1; and wherein saidliquid nutrient medium comprises one or more of the following: Fe, PO₄,Zn, Mn, B, Co, Cu, Ni, Mo, Se, W, K, Na, Mg, Cl, and S.
 2. The method ofclaim 1, wherein said vitamin solution comprises one or more of biotin,thiamine, and pantothenic acid.
 3. The method of claim 1, wherein thefermentation broth includes calcium pantothenate and cobalt at a ratioof calcium pantothenate to cobalt in a range of 0.4-0.5.
 4. The methodof claim 1, wherein the strain of Clostridium ljungdahlii is selectedfrom the strains consisting of Clostridium ljungdahlii PETC, Clostridiumljungdahlii ER12, Clostridium ljungdahlii C-01, and Clostridiumljungdahlii O-52.
 5. The method of claim 1, wherein the ratio of ethanolto acetate and the ratio of acetic acid to ethanol is measured after thebacteria achieve a stable cell concentration in the bioreactor.
 6. Themethod of claim 1, wherein said gaseous substrate is selected from thegroup consisting of (a) carbon monoxide, (b) carbon monoxide andhydrogen, and (c) carbon monoxide, carbon dioxide and hydrogen.
 7. Themethod of claim 1, wherein the gaseous substrate additionally comprisesat least one gas chosen from nitrogen and methane.
 8. The method ofclaim 1 further comprising supplying calcium pantothenate in an amountranging from 2 to 50 μg calcium pantothenate per gram of dry cells ofbacteria in the fermentation bioreactor.
 9. The method of claim 1,wherein cobalt is provided in an amount ranging from 5 to 100 μg cobaltper gram of dry cells of bacteria in the fermentation bioreactor. 10.The method or claim 1, wherein cobalt is provided in an amount rangingfrom 20 to 50 μg cobalt per gram of dry cells of bacteria in thefermentation bioreactor.
 11. The method of claim 1, wherein cobalt ismaintained at a constant concentration.
 12. The method of claim 1,wherein the fermentation bioreactor is selected from the groupconsisting of one or more continuous stirred tank reactor (CSTR),immobilized cell reactor (ICR), trickle bed reactor (TBR), bubblecolumn, gas lift fermenter, and static mixer.
 13. The method of claim 1,wherein the fermentation bioreactor comprises a growth reactor thatfeeds the fermentation broth to a second fermentation bioreactor inwhich some or all of the ethanol is produced.
 14. The method of claim 1further comprising purging the bacteria from the fermentationbioreactor.
 15. The method of claim 1, wherein the pH is 4.5 to 5.5.